U.S. patent application number 12/535468 was filed with the patent office on 2010-02-18 for 2-amino pyrimidine compounds as potent hsp-90 inhibitors.
This patent application is currently assigned to Pfizer Inc. Invention is credited to Pei-Pei Kung, Jerry Jialun Meng.
Application Number | 20100041681 12/535468 |
Document ID | / |
Family ID | 41151854 |
Filed Date | 2010-02-18 |
United States Patent
Application |
20100041681 |
Kind Code |
A1 |
Kung; Pei-Pei ; et
al. |
February 18, 2010 |
2-AMINO PYRIMIDINE COMPOUNDS AS POTENT HSP-90 INHIBITORS
Abstract
The present invention is directed to compounds of formula (I),
##STR00001## or pharmaceutically acceptable salts thereof, their
synthesis, and their use as HSP-90 inhibitors.
Inventors: |
Kung; Pei-Pei; (San Diego,
CA) ; Meng; Jerry Jialun; (San Diego, CA) |
Correspondence
Address: |
PFIZER INC
10555 SCIENCE CENTER DRIVE
SAN DIEGO
CA
92121
US
|
Assignee: |
Pfizer Inc
|
Family ID: |
41151854 |
Appl. No.: |
12/535468 |
Filed: |
August 4, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61088599 |
Aug 13, 2008 |
|
|
|
Current U.S.
Class: |
514/265.1 ;
435/375; 544/280 |
Current CPC
Class: |
A61P 35/00 20180101;
C07D 487/04 20130101 |
Class at
Publication: |
514/265.1 ;
544/280; 435/375 |
International
Class: |
A61K 31/519 20060101
A61K031/519; C07D 487/04 20060101 C07D487/04; C12N 5/02 20060101
C12N005/02; A61P 35/04 20060101 A61P035/04 |
Claims
1. A compound of formula (I) ##STR00054## or a pharmaceutically
acceptable salt thereof, wherein: R.sup.1 is selected from the
group consisting of C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkyl
substituted with 1 to 6 fluorine, C.sub.1-C.sub.6 alkyl substituted
with 1 to 2 chlorine and up to six fluorine, C.sub.3-C.sub.8
cycloalkyl, and C.sub.3-C.sub.8 cycloalkyl substituted with 1 to 6
groups selected from fluorine, chlorine and C.sub.1-C.sub.3 alkyl;
R.sup.2 and R.sup.3 are independently selected from the group
consisting of chlorine, fluorine, --CN, C.sub.1-C.sub.3 alkoxyl,
C.sub.1-C.sub.3 alkyl and C.sub.1-C.sub.3 alkyl substituted with 1
to 6 fluorine; R.sup.4 is selected from the group consisting of
hydrogen, fluorine, chlorine, bromine, --CN, C.sub.1-C.sub.3 alkyl
and C.sub.1-C.sub.3 alkyl substituted with 1 to 6 fluorine; R.sup.5
and R.sup.6 are independently selected from the group consisting of
hydrogen, fluorine, chlorine, bromine, --CN, C.sub.1-C.sub.3 alkyl
and C.sub.1-C.sub.3 alkyl substituted with 1 to 6 fluorine;
provided that (1) when R.sup.4, R.sup.5 and R.sup.6 are all
hydrogen, then R.sup.2 and R.sup.3 are not both chlorine; and (2)
the compound of formula I is not any of the following compounds,
##STR00055##
2. The compound or salt of claim 1, wherein R.sup.4 is hydrogen,
R.sup.5 is hydrogen, R.sup.6 is hydrogen, and R.sup.3 is selected
from the group consisting of fluorine, C.sub.1-C.sub.3 alkyl and
C.sub.1-C.sub.3 alkyl substituted with 1 to 6 fluorine.
3. The compound or salt of claim 1, wherein R.sup.4 is hydrogen,
R.sup.5 is hydrogen, R.sup.6 is hydrogen, and R.sup.2 is selected
from the group consisting of fluorine, C.sub.1-C.sub.3 alkyl and
C.sub.1-C.sub.3 alkyl substituted with 1 to 6 fluorine.
4. The compound or salt of claim 1, wherein R.sup.4 is selected
from fluorine, chlorine, C.sub.2-C.sub.3 alkyl and C.sub.1-C.sub.3
alkyl substituted with 1 to 6 fluorine.
5. The compound or salt of claim 1, wherein R.sup.4 is fluorine or
chlorine, R.sup.5 is hydrogen, and R.sup.6 is hydrogen.
6. The compound or salt of claim 1, wherein R.sup.4 is fluorine or
chlorine, R.sup.5 is hydrogen, R.sup.6 is hydrogen, R.sup.2 is
methyl or chlorine, and R.sup.3 is methyl or chlorine.
7. The compound or salt of claim 1, wherein R.sup.5 is
C.sub.1-C.sub.3 alkyl substituted with 1 to 6 fluorine.
8. The compound or salt of claim 1, wherein R.sup.6 is
C.sub.1-C.sub.3 alkyl substituted with 1 to 6 fluorine.
9. The compound or salt of claim 1, wherein R.sup.1 is selected
from the group consisting of cyclopropyl, cyclobutyl,
bicyclo[1.1.1]pent-1-yl, and C.sub.1-C.sub.6 alkyl substituted with
1 to 6 fluorine.
10. The compound or salt of claim 1, wherein R.sup.1 is
C.sub.3-C.sub.8 cycloalkyl or C.sub.3-C.sub.8 cycloalkyl
substituted with 1 to 6 groups selected from fluorine, chlorine and
C.sub.1-C.sub.3 alkyl.
11. The compound or salt of claim 1, wherein R.sup.1 is
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkyl substituted with 1 to
6 fluorine, or C.sub.1-C.sub.6 alkyl substituted with 1 to 2
chlorine and up to six fluorine.
12. The compound or salt of claim 1, wherein R.sup.2 is chlorine
and R.sup.3 is chlorine.
13. The compound or salt of claim 1, wherein R.sup.2 is chlorine
and R.sup.3 is C.sub.1-C.sub.3 alkyl.
14. A compound selected from the group consisting of ##STR00056##
##STR00057## ##STR00058## ##STR00059## or a pharmaceutically
acceptable salt thereof.
15. A compound of the formula ##STR00060## or a pharmaceutically
acceptable salt thereof.
16. A pharmaceutical composition comprising the compound of claim
1, or a pharmaceutically acceptable salt thereof, and a
pharmaceutically acceptable carrier.
17. The use of the compound of claim 1, or a pharmaceutically
acceptable salt thereof, in the preparation of a medicament for the
treatment of cancer.
18. A method of modulating the activity of HSP-90, comprising
contacting a cell with a compound of claim 1, or a pharmaceutically
acceptable salt thereof.
Description
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 61/088,599, filed Aug. 13, 2008, the
disclosure of which is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention is directed to compounds, and
pharmaceutically acceptable salts and solvates thereof, their
synthesis, and their use as modulators or inhibitors of HSP-90. The
compounds of the present invention are useful for modulating (e.g.
inhibiting) HSP-90 activity and for treating diseases or conditions
mediated by HSP-90, such as for example, disease states associated
with abnormal cell growth such as cancer.
BACKGROUND
[0003] Molecular chaperones play important roles in cellular
function by ensuring proper folding of proteins upon synthesis as
well as their refolding under conditions of denaturing stress. By
regulating the balance between protein synthesis and degradation,
molecular chaperones are a significant part of the cellular
response to stress. In addition, by regulating the proper folding
of various cellular proteins, chaperones play an important role in
regulating cellular functions such as cell proliferation and
apoptosis. (See, e.g. Jolly, et al., J. Natl. Cancer Inst. 92:
1564-1572 (2000)). Heat shock proteins (HSPs) are a class of
chaperones that accumulate in the cell in response to various
environmental stresses, such as heat shock, oxidative stress, or
the presence of alcohols or heavy metals. In addition to their role
in protecting the cell from such environmental stresses, HSPs may
also play a significant role as chaperones for a variety of
cellular proteins under stress-free conditions. Members of the HSP
family are classified according to their molecular weight (e.g.
HSP-27, HSP-70, and HSP-90). Evidence of differential expression of
HSPs in various stages of tumor progression suggests HSPs play a
role in cancer. (See, e.g. Martin, et al., Cancer Res. 60:2232-2238
(2000)).
[0004] HSP-90 is a homodimer with ATPase activity and functions in
a series of complex interactions with a variety of substrate
proteins (Young, et al., J. Cell Biol. 154: 267-273 (2001)). HSP-90
is unique with regard to other chaperones, however, since most of
its known substrate proteins are signal transduction proteins.
Thus, HSP-90 plays an essential role in regulating cellular signal
transduction networks. (See, e.g. Xu, et al., Proc. Natl. Acad.
Sci. 90:7074-7078 (1993)). In particular, substrate proteins of
HSP-90 include many mutated or over-expressed proteins implicated
in cancer such as p53, Bcr-Abl kinase, Raf-1 kinase, Akt kinase,
Npm-Alk kinase p185.sup.ErbB2 transmembrane kinase, Cdk4, Cdk6,
Wee1 (a cell cycle-dependent kinase), HER2/Neu (ErbB2), and hypoxia
inducible factor-1.alpha. (HIF-1.alpha.). Thus inhibition of HSP-90
results in selective degradation of these important signaling
proteins involved in apoptosis, cell proliferation, and cell cycle
regulation (Holstein, et al., Cancer Res. 61:4003-4009 (2001)).
Accordingly, HSP-90 is an attractive therapeutic target because of
the important roles played by these signaling proteins in disease
states involving abnormal cell growth, such as cancer. It is thus
desirable to discover and develop new inhibitors of HSP-90 activity
that can provide a therapeutic benefit to patients suffering from
disease states related to abnormal cell growth such as cancer.
SUMMARY
[0005] In one embodiment, the invention provides a compound of
formula (I),
##STR00002##
or a pharmaceutically acceptable salt thereof, wherein:
[0006] R.sup.1 is selected from the group consisting of
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkyl substituted with 1 to
6 fluorine, C.sub.1-C.sub.6 alkyl substituted with 1 to 2 chlorine
and up to six fluorine, C.sub.3-C.sub.8 cycloalkyl, and
C.sub.3-C.sub.8 cycloalkyl substituted with 1 to 6 groups
independently selected from fluorine, chlorine and C.sub.1-C.sub.3
alkyl;
[0007] R.sup.2 and R.sup.3 are each independently selected from the
group consisting of chlorine, fluorine, C.sub.1-C.sub.3 alkyl and
C.sub.1-C.sub.3 alkyl substituted with 1 to 6 fluorine;
[0008] R.sup.4 is selected from the group consisting of hydrogen,
fluorine, chlorine, bromine, --CN, C.sub.1-C.sub.3 alkyl and
C.sub.1-C.sub.3 alkyl substituted with 1 to 6 fluorine;
[0009] R.sup.5 and R.sup.6 are each independently selected from the
group consisting of hydrogen, fluorine, chlorine, bromine, --CN,
C.sub.1-C.sub.3 alkyl and C.sub.1-C.sub.3 alkyl substituted with 1
to 6 fluorine;
[0010] provided that
[0011] (1) when R.sup.4, R.sup.5 and R.sup.6 are all hydrogen, then
R.sup.2 and R.sup.3 are not both chlorine; and
[0012] (2) the compound of formula I is not any of the following
compounds,
##STR00003##
[0013] In a preferred aspect of the embodiment, and in combination
with any other preferred aspects not inconsistent, R.sup.4 is
hydrogen, R.sup.5 is hydrogen, R.sup.6 is hydrogen, R.sup.3 is
selected from the group consisting of fluorine, C.sub.1-C.sub.3
alkyl and C.sub.1-C.sub.3 alkyl substituted with 1 to 6 fluorine.
Preferably, R.sup.3 is C.sub.1-C.sub.3 alkyl.
[0014] In another preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.4 is hydrogen, R.sup.5 is hydrogen,
[0015] R.sup.6 is hydrogen, and R.sup.2 is selected from the group
consisting of fluorine, C.sub.1-C.sub.3 alkyl and C.sub.1-C.sub.3
alkyl substituted with 1 to 6 fluorine.
[0016] In another preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.4 is selected from fluorine, chlorine, C.sub.2-C.sub.3 alkyl
and C.sub.1-C.sub.3 alkyl substituted with 1 to 6 fluorine.
Preferably, R.sup.4 is selected from fluorine and chlorine. More
preferably, R.sup.4 is fluorine.
[0017] In another preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.4 is fluorine or chlorine, R.sup.5 is hydrogen, and R.sup.6
is hydrogen. Preferably, R.sup.4 is fluorine, and R.sup.5 and
R.sup.6 are both hydrogen.
[0018] In another preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.4 is fluorine or chlorine, R.sup.5 is hydrogen, R.sup.6 is
hydrogen, R.sup.2 is methyl or chlorine, and R.sup.3 is methyl or
chlorine.
[0019] In another preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.5 is C.sub.1-C.sub.3 alkyl substituted with 1 to 6
fluorine.
[0020] In another preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.6 is C.sub.1-C.sub.3 alkyl substituted with 1 to 6
fluorine.
[0021] In another preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.1 is selected from the group consisting of cyclopropyl,
cyclobutyl, bicyclo[1.1.1]pent-1-yl, and C.sub.1-C.sub.6 alkyl
substituted with 1 to 6 fluorine.
[0022] In another preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.1 is C.sub.3-C.sub.8 cycloalkyl or C.sub.3-C.sub.8 cycloalkyl
substituted with 1 to 6 groups selected from fluorine, chlorine and
C.sub.1-C.sub.3 alkyl.
[0023] In another preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.1 is C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkyl substituted
with 1 to 6 fluorine, or C.sub.1-C.sub.6 alkyl substituted with 1
to 2 chlorine and up to 6 fluorine.
[0024] In another preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.2 is chlorine and R.sup.3 is chlorine.
[0025] In another preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.2 is chlorine and R.sup.3 is C.sub.1-C.sub.3 alkyl. In
another preferred aspect of the embodiment, and in combination with
any other preferred aspects not inconsistent, R.sup.4 is fluorine
or chlorine, R.sup.5 is hydrogen, R.sup.6 is hydrogen, R.sup.2 is
methyl or chlorine, R.sup.3 is methyl or chlorine, R.sup.1 is
selected from the group consisting of cyclopropyl, cyclobutyl,
bicyclo[1.1.1]pent-1-yl, and C.sub.1-C.sub.6 alkyl substituted with
1 to 6 fluorine. Preferably, R.sup.4 is fluorine, R.sup.2 is
chlorine and R.sup.3 is chlorine. More preferably, R.sup.4 is
fluorine, R.sup.2 is chlorine and R.sup.3 is methyl.
[0026] In another embodiment, the invention provides a compound of
formula I,
##STR00004##
wherein:
[0027] R.sup.1 is selected from the group consisting of
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkyl substituted with 1 to
6 fluorine, C.sub.1-C.sub.6 alkyl substituted with 1 to 2 chlorine
and up to 6 fluorine, C.sub.3-C.sub.8 cycloalkyl, and
C.sub.3-C.sub.8 cycloalkyl substituted with 1 to 6 groups
independently selected from fluorine, chlorine and C.sub.1-C.sub.3
alkyl;
[0028] R.sup.2 and R.sup.3 are each independently selected from the
group consisting of chlorine, fluorine, C.sub.1-C.sub.3 alkyl and
C.sub.1-C.sub.3 alkyl substituted with 1 to 6 fluorine;
[0029] R.sup.4 is selected from fluorine, chlorine, C.sub.2-C.sub.3
alkyl and C.sub.1-C.sub.3 alkyl substituted with 1 to 6 fluorine.
Preferably, R.sup.4 is selected from fluorine and chlorine. More
preferably, R.sup.4 is fluorine.
[0030] R.sup.5 and R.sup.6 are each independently selected from the
group consisting of hydrogen, fluorine, chlorine, bromine, --CN,
C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3 alkyl substituted with 1 to
6 fluorine;
or a pharmaceutically acceptable salt thereof.
[0031] In one preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.4 is fluorine or chlorine, R.sup.5 is hydrogen, and R.sup.6
is hydrogen. More preferably, R.sup.4 is fluorine, and R.sup.5 and
R.sup.6 are both hydrogen.
[0032] In another preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.4 is fluorine or chlorine, R.sup.5 is hydrogen, R.sup.6 is
hydrogen, R.sup.2 is methyl or chlorine, and R.sup.3 is methyl or
chlorine.
[0033] In another preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.5 is C.sub.1-C.sub.3 alkyl substituted with 1 to 6
fluorine.
[0034] In another preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.6 is C.sub.1-C.sub.3 alkyl substituted with 1 to 6
fluorine.
[0035] In another preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.1 is selected from the group consisting of cyclopropyl,
cyclobutyl, bicyclo[1.1.1]pent-1-yl, and C.sub.1-C.sub.6 alkyl
substituted with 1 to 6 fluorine.
[0036] In another preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.1 is C.sub.3-C.sub.8 cycloalkyl or C.sub.3-C.sub.8 cycloalkyl
substituted with 1 to 6 groups independently selected from
fluorine, chlorine and C.sub.1-C.sub.3 alkyl.
[0037] In another preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.1 is C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkyl substituted
with 1 to 6 fluorine, C.sub.1-C.sub.6 alkyl substituted with 1 to 2
chlorine and up to six fluorine.
[0038] In another preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.2 is chlorine and R.sup.3 is chlorine.
[0039] In another preferred aspect of the embodiment, and in
combination with any other preferred aspects not inconsistent,
R.sup.2 is chlorine and R.sup.3 is C.sub.1-C.sub.3 alkyl. In
another preferred aspect of the embodiment, and in combination with
any other preferred aspects not inconsistent, R.sup.4 is fluorine
or chlorine, R.sup.5 is hydrogen, R.sup.6 is hydrogen, R.sup.2 is
methyl or chlorine, R.sup.3 is methyl or chlorine and, R.sup.1 is
selected from the group consisting of cyclopropyl, cyclobutyl,
bicyclo[1.1.1]pent-1-yl, and C.sub.1-C.sub.6 alkyl substituted with
1 to 6 fluorine. Preferably, R.sup.4 is fluorine, R.sup.2 is
chlorine and R.sup.3 is chlorine. More preferably, R.sup.4 is
fluorine, R.sup.2 is chlorine and R.sup.3 is methyl.
[0040] In another embodiment, the invention provides a compound
selected from the group consisting of
##STR00005## ##STR00006## ##STR00007## ##STR00008##
or a pharmaceutically acceptable salt thereof.
[0041] In another embodiment, the present invention provides a
pharmaceutical composition comprising a compound of formula I, or a
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier.
[0042] In another embodiment, the present invention provides a use
of a compound of formula I or a pharmaceutically acceptable salt
thereof, in the preparation of a medicament.
[0043] In another embodiment, the present invention provides a use
of a compound of formula I or a pharmaceutically acceptable salt
thereof, in the preparation of a medicament for the treatment of
cancer.
[0044] In another embodiment, the present invention provides a
method of modulating the activity of HSP-90, comprising contacting
a cell with a compound of formula I or a pharmaceutically
acceptable salt thereof.
[0045] In another embodiment, the invention provides a combination
of the compound or salt of Formula I and a further therapeutic
agent. In one aspect of this embodiment, the combination is used
for the treatment of abnormal cell growth, preferably cancer.
[0046] This invention also relates to a method for the treatment of
abnormal cell growth in a mammal, including a human, comprising
administering to said mammal an amount of a compound of the Formula
I, as defined above, or a pharmaceutically acceptable salt or
solvate thereof, that is effective in treating abnormal cell
growth.
[0047] In one embodiment of this method, the abnormal cell growth
is cancer, including, but not limited to, mesothelioma,
hepatobilliary (hepatic and billiary duct), a primary or secondary
CNS tumor, a primary or secondary brain tumor, lung cancer (NSCLC
and SCLC), bone cancer, pancreatic cancer, skin cancer, cancer of
the head or neck, cutaneous or intraocular melanoma, ovarian
cancer, colon cancer, rectal cancer, cancer of the anal region,
stomach cancer, gastrointestinal (gastric, colorectal, and
duodenal), breast cancer, uterine cancer, carcinoma of the
fallopian tubes, carcinoma of the endometrium, carcinoma of the
cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's
Disease, cancer of the esophagus, cancer of the small intestine,
cancer of the endocrine system, cancer of the thyroid gland, cancer
of the parathyroid gland, cancer of the adrenal gland, sarcoma of
soft tissue, cancer of the urethra, cancer of the penis, prostate
cancer, testicular cancer, chronic or acute leukemia, chronic
myeloid leukemia, lymphocytic lymphomas, cancer of the bladder,
cancer of the kidney or ureter, renal cell carcinoma, carcinoma of
the renal pelvis, neoplasms of the central nervous system (CNS),
primary CNS lymphoma, non-hodgkin's lymphoma, spinal axis tumors,
brain stem glioma, pituitary adenoma, adrenocortical cancer, gall
bladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma,
neuroblastoma, and retinoblastoma, or a combination of one or more
of the foregoing cancers.
[0048] In another embodiment of said method, said abnormal cell
growth is a benign proliferative disease, including, but not
limited to, psoriasis, benign prostatic hypertrophy and
restinosis.
[0049] In a preferred embodiment of the present invention the
cancer is selected from lung cancer (NSCLC and SCLC), cancer of the
head or neck, ovarian cancer, colon cancer, rectal cancer, cancer
of the anal region, stomach cancer, breast cancer, cancer of the
kidney or ureter, renal cell carcinoma, carcinoma of the renal
pelvis, neoplasms of the central nervous system (CNS), primary CNS
lymphoma, non-hodgkin's lymphoma, and spinal axis tumors, or a
combination of one or more of the foregoing cancers.
[0050] In another preferred embodiment of the present invention the
cancer is selected from lung cancer (NSCLC and SCLC), ovarian
cancer, colon cancer, rectal cancer, and cancer of the anal region,
or a combination of one or more of the foregoing cancers.
[0051] In a more preferred embodiment of the present invention the
cancer is selected from lung cancer (NSCLC and SCLC), ovarian
cancer, colon cancer, and rectal cancer, or a combination of one or
more of the foregoing cancers.
[0052] In another embodiment of said method, said abnormal cell
growth is a benign proliferative disease, including, but not
limited to, psoriasis, benign prostatic hypertrophy and
restinosis.
[0053] This invention also relates to a pharmaceutical composition
for the treatment of abnormal cell growth in a mammal, including a
human, comprising an amount of a compound of the formula I, as
defined above, or a pharmaceutically acceptable salt thereof, that
is effective in treating abnormal cell growth, and a
pharmaceutically acceptable carrier. In one embodiment of said
composition, said abnormal cell growth is cancer, including, but
not limited to, mesothelioma, hepatobilliary (hepatic and billiary
duct), a primary or secondary CNS tumor, a primary or secondary
brain tumor, lung cancer (NSCLC and SCLC), bone cancer, pancreatic
cancer, skin cancer, cancer of the head or neck, cutaneous or
intraocular melanoma, ovarian cancer, colon cancer, rectal cancer,
cancer of the anal region, stomach cancer, gastrointestinal
(gastric, colorectal, and duodenal), breast cancer, uterine cancer,
carcinoma of the fallopian tubes, carcinoma of the endometrium,
carcinoma of the cervix, carcinoma of the vagina, carcinoma of the
vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the
small intestine, cancer of the endocrine system, cancer of the
thyroid gland, cancer of the parathyroid gland, cancer of the
adrenal gland, sarcoma of soft tissue, cancer of the urethra,
cancer of the penis, prostate cancer, testicular cancer, chronic or
acute leukemia, chronic myeloid leukemia, lymphocytic lymphomas,
cancer of the bladder, cancer of the kidney or ureter, renal cell
carcinoma, carcinoma of the renal pelvis, neoplasms of the central
nervous system (CNS), primary CNS lymphoma, non hodgkin's lymphoma,
spinal axis tumors, brain stem glioma, pituitary adenoma,
adrenocortical cancer, gall bladder cancer, multiple myeloma,
cholangiocarcinoma, fibrosarcoma, neuroblastoma, and
retinoblastoma, or a combination of one or more of the foregoing
cancers. In another embodiment of said pharmaceutical composition,
said abnormal cell growth is a benign proliferative disease,
including, but not limited to, psoriasis, benign prostatic
hypertrophy and restinosis.
[0054] As used herein, the symbol [------] when incorporated into
the chemical structure of a substituent means that the atom to
which [------] is attached is the point of attachment of that
substitutent to some position on another molecule. For example, X
in the hypothetical molecule CH.sub.3CH.sub.2--X might be defined
as X is
##STR00009##
In which case, the placement of [------] attached to the
arbitrarily numbered position C-1, means that C-1 of the phenyl
ring is attached to the methylene carbon.
[0055] "C.sub.m-C.sub.n alkyl", wherein m is an integer of 1-19, n
is an integer of 2 to 20 and n>m, refers to a straight chain or
branched saturated hydrocarbon radical having from m to n carbon
atoms, wherein n is an integer of 2 to 20. Examples of
C.sub.m-C.sub.n alkyl groups include, but are not limited to
methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, iso-butyl,
tert-butyl, pentyl, neo-pentyl, sec-pentyl, hexyl, heptyl, octyl,
and the like, including substituted forms thereof. Further, the
term "alkyl" refers to a straight chain or branched saturated
hydrocarbon radical of 1 to 20 carbon atoms, or 1 to 12 carbon
atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 4
carbon atoms. Alkyl may be may be unsubstituted or further
substituted by at least one substituent.
[0056] "C.sub.m-C.sub.n cycloalkyl", wherein m is an integer of
3-19, n is an integer of 4 to 20 and n>m, refers to a cyclic
saturated hydrocarbon radical having from m to n carbon atoms. A
cycloalkyl group may be monocyclic and where permissible may be
bicyclic or polycyclic. A cycloalkyl may also be a spirocyclic.
Illustrative examples of cycloalkyl are derived from, but not
limited to, the following:
##STR00010##
[0057] "C.sub.m-C.sub.n Alkoxy" or "C.sub.m-C.sub.n alkoxyl" refers
to --O--(C.sub.m-C.sub.n alkyl) Wherein (C.sub.m-C.sub.n alkyl) is
as defined previously in this section.
[0058] "Amino" refers to --NH.sub.2.
[0059] When a group is "optionally substituted" or "optionally
further substituted" by some substituents, it means a carbon or a
nitrogen atom of this group wherein one or more hydrogen atoms are
attached to the carbon or nitrogen atom, such carbon or nitrogen
atom is optionally substituted by some other substituents. For
example, "R is H, C.sub.1-C.sub.3 alkyl or phenyl, and R is
optionally further substituted by 1-3 groups selected from --F, oxo
and C.sub.1-C.sub.3 perfluoroalkyl", means that R is 1) H (when R
is H, R cannot be further substituted); 2) C.sub.1-C.sub.3 alkyl
optionally further substituted by 1-3 groups selected from --F, oxo
and C.sub.1-C.sub.3 perfluoroalkyl; and 3) phenyl optionally
further substituted by 1-3 groups selected from --F and
C.sub.1-C.sub.3 perfluoroalkyl. Optional substitution of phenyl by
oxo does not apply when R is phenyl because no single atom of the
phenyl group possess two hydrogen atoms to be substituted by oxo,
i.e. .dbd.O bond. When a group is further substituted by a
"-(C.sub.1-C.sub.4 alkylene)-", it means the "-(C.sub.1-C.sub.4
alkylene)-", together with the nitrogen atom or the carbon atom of
the group to which "C.sub.1-C.sub.4 alkylene" is attached, form a
carbo or hetero spirocycle.
[0060] A "pharmaceutical composition" refers to a mixture of one or
more of the compounds described herein, or
physiologically/pharmaceutically acceptable salts, solvates,
hydrates or prodrugs thereof, with other chemical components, such
as physiologically/pharmaceutically acceptable carriers and
excipients. The purpose of a pharmaceutical composition is to
facilitate administration of a compound to an organism, such as a
mammal, including a human.
[0061] As used herein, a "physiologically/pharmaceutically
acceptable carrier" refers to a carrier or diluent that does not
cause significant irritation to an organism such as a mammal,
including a human and does not abrogate the biological activity and
properties of the administered compound.
[0062] A "pharmaceutically acceptable excipient" refers to an inert
substance added to a pharmaceutical composition to further
facilitate administration of a compound. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0063] As used herein, the term "pharmaceutically acceptable salt"
refers to those salts that retain the biological effectiveness and
properties of the parent compound. Such salts include:
[0064] (1) acid addition salts, which can be obtained by reaction
of the free base of the parent compound with inorganic acids such
as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric
acid, sulfuric acid, and perchloric acid and the like, or with
organic acids such as acetic acid, oxalic acid, (D) or (L) malic
acid, maleic acid, methanesulfonic acid, ethanesulfonic acid,
p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid,
succinic acid or malonic acid and the like; or
[0065] (2) salts formed when an acidic proton present in the parent
compound either is replaced by a metal ion, e.g., an alkali metal
ion, an alkaline earth ion, or an aluminum ion; or coordinates with
an organic base such as ethanolamine, diethanolamine,
triethanolamine, tromethamine, N-methylglucamine, and the like.
[0066] "Contacting" refers to bringing a compound of the present
teachings and a target PK together in such a manner that the
compound can affect the catalytic activity of the PK, either
directly, i.e., by interacting with the kinase itself, or
indirectly, i.e., by interacting with another molecule on which the
catalytic activity of the kinase is dependent. Such "contacting"
can be accomplished "in vitro," i.e., in a test tube, a petri dish
or the like. In a test tube, contacting may involve only a compound
and a PK of interest or it may involve whole cells. Cells may also
be maintained or grown in cell culture dishes and contacted with a
compound in that environment. In this context, the ability of a
particular compound to affect a PK related disorder, i.e., the
IC.sub.50 of the compound, can be determined. For cells outside a
organism, multiple methods exist that are known to those skilled in
the art, to get the PKs in contact with the compounds including,
but not limited to, direct cell microinjection and numerous
transmembrane carrier techniques.
[0067] "PK related disorder," "PK driven disorder," and "abnormal
PK activity" all refer to a condition characterized by
inappropriate, i.e., under or, over, PK catalytic activity, where
the particular PK can be an RTK, a CTK or an STK. Inappropriate
catalytic activity can arise from: (1) PK expression in cells which
normally do not express PKs, (2) increased PK expression leading to
unwanted cell proliferation, differentiation and/or growth, or, (3)
decreased PK expression leading to unwanted reductions in cell
proliferation, differentiation and/or growth. Over-activity of a PK
refers to either amplification of the gene encoding a particular PK
or production of a level of PK activity which can correlate with a
cell proliferation, differentiation and/or growth disorder (that
is, as the level of the PK increases, the severity of one or more
of the symptoms of the cellular disorder increases). Under-activity
is, wherein the severity of one or more symptoms of a cellular
disorder increase as the level of the PK activity decreases.
[0068] "Treat", "treating" and "treatment" refer to a method of
alleviating or abrogating a PK mediated cellular disorder and/or
its attendant symptoms. With regard to cancer, these terms mean
that the life expectancy of an individual affected with a cancer
will be increased or that one or more of the symptoms of the
disease will be reduced.
[0069] "Organism" refers to any living entity comprised of at least
one cell. A living organism can be as simple as, for example, a
single eukariotic cell or as complex as a mammal, including a human
being.
[0070] "Therapeutically effective amount" refers to that amount of
the compound being administered which will relieve to some extent
one or more of the symptoms of the disorder being treated. In
reference to the treatment of cancer, a therapeutically effective
amount refers to that amount which has at least one of the
following effects:
[0071] (1) reducing the size of the tumor;
[0072] (2) inhibiting (that is, slowing to some extent, preferably
stopping) tumor metastasis;
[0073] (3) inhibiting to some extent (that is, slowing to some
extent, preferably stopping) tumor growth, and
[0074] (4) relieving to some extent (or, preferably, eliminating)
one or more symptoms associated with the cancer.
[0075] The term "stereoisomers" refers to compounds that have
identical chemical constitution, but differ with regard to the
arrangement of their atoms or groups in space. In particular, the
term "enantiomers" refers to two stereoisomers of a compound that
are non-superimposable mirror images of one another. The terms
"racemic" or "racemic mixture," as used herein, refer to a 1:1
mixture of enantiomers of a particular compound. The term
"diastereomers", on the other hand, refers to the relationship
between a pair of stereoisomers that comprise two or more
asymmetric centers and are not mirror images of one another.
DETAILED DESCRIPTION
[0076] The compounds of the current invention, i.e., the compounds
of formula I, can be made following the method of Examples 1-16.
The following reaction Schemes 1-3 will enable one of ordinary
skill in the art to make the compound of formula I.
##STR00011##
##STR00012##
##STR00013##
[0077] Scheme 1 illustrates the synthesis of intermediate I(C) used
to make compounds of formula I. The beta keto ester I(A) can be
prepared based on a known procedures (see, e.g. Viscontini and
Buhler Helvetica Chimica Acta, 50(5): 1289-93; (1967), Rosowsky et.
al. J. Heterocyclic Chem., 26: 509-16 (1989)). PG.sup.1, the
nitrogen protecting group, can be selected for compatability with
subsequent chemistry. Protecting groups and general considerations
for their use are described in T. Greene and P. Wuts, "Protective
Groups in Organic Synthesis", 3.sup.rd Edition 1999, John Wiley
& Sons and are well known to those skilled in the art. Compound
I(A) is condensed with guanidine to give compound I(B). This can
typically be done by heating compound I(A) with guanidine or
guanidine equivalent a protic solvent. A typical reaction condition
would be to reflux compound I(A) with guandidine carbonate in
tert-butanol as a solvent. Conversion of the hydroxyl group of
compound I(B) to chloro or iodo gives I(C). This can typically be
done by heating compound I(B) with POCl.sub.3 in an aprotic
solvent. A typical reaction condition would be to reflux compound
I(B) together with excess POCl.sub.3 either neat or in dry
acetonitrile as solvent.
[0078] Scheme 2 illustrates the route through which compounds of
formula I can be made from intermediate I(C). In Scheme 2, the
chloro/iodo group of compound I(C) is replaced by a trisubstituted
phenyl group to give compound II(A). The displacement of the chloro
leaving group of compound I(C) by the trisubstituted phenyl group
to give compound II(A) can be carried out using cross coupling
methodology utilizing Suzuki, Stille, Negishi or similar
conditions. A typical cross coupling reaction for the
transformation of compound I(C) to compound II(A) would be to treat
compound I(C) with a boronic acid or ester in the presence of a
base such as sodium carbonate and Pd(0) catalyst in a solvent
mixture such as water and 1,4-dioxane to give compound II(A). The
nitrogen protecting group, PG.sup.1, of compound II(A) is then
removed to give compound II(B). When PG.sup.1 forms an ethyl
carbamate protecting group, removing of PG.sup.1 can typically be
done, by refluxing compound II(A) with trimethylsilyliodide in a
solvent such as CH.sub.3CN. Alternatively HBr in acetic acid or KOH
in isopropanol can also be used to remove the ethyl carbamate
protecting group. When PG.sup.1 is a tert-butyl carbamate
protecting group, removing of PG.sup.1 can be done by treating
compound II(A) with hydrogen chloride in a solvent such as
1,4-dioxane. A typical condition for the transformation of compound
II(A) to compound II(B) is to treat compound II(A) with TMSI (10-20
equivalents, 4M in 1,4-dioxane) to give compound II(B). The
dihydropyrrolo amino moiety of compound II(B) then acts as a
nucleophile in reactions with an electrophilic R.sup.1--NH--CO
moiety to give compound 1. This nucleophilic reaction can be
acylation and other reactions applicable to secondary alkyl amines.
A typical acylation reaction condition is to react compound II(B)
with an R.sup.1 isocyanate or isocyanate equivalent moiety in the
presence of TEA to give compound I as a urea. A method to prepare
isocyanate equivalent is to form a adduct of CDI
(1,1'-carbonyldiimidazole) with different amines. The CDI adduct
can then react with compound II(B) to give compound I in the
presence of TEA in DMF at elevated temperature.
[0079] Scheme 3 illustrates another route through which compounds
of formula I can be made from intermediate I(C). In Scheme 3 when
PG.sup.1 is an ethyl carbamate protecting group, PG.sup.1 of
compound I(C) is removed with concurrent conversion of chloro to
iodo in one step. This can typically be carried out by treating
compound I(C) with TMSI at elevated temperature or with hydrogen
chloride at room temperature in an aprotic solvent. A typical
reaction condition is to reflux compound I(C) in CH.sub.3CN with
five equivalents of TMSI. Following a methanol quench, compound
III(A) is obtained as an HI salt. The dihydropyrrolo amino moiety
of compound III(A) then reacts, as a nucleophile, with an
electrophilic R.sup.1 moiety to give compound III(B). This
nucleophilic reaction can be acylation and other reactions
applicable to secondary alkyl amines. A typical acylation reaction
condition is to react compound III(A) with an R.sup.1 isocyanate or
isocyanate equivalent moiety in the presence of TEA to give
compound III(B) as a urea. A method to prepare isocyanate
equivalent is to form a adduct of CDI (1,1'-carbonyldiimidazole)
with different amines. The CDI adduct can then react with compound
II(B) to give compound I in the presence of TEA in DMF at elevated
temperature. The iodo group of compound III(B) is then displaced by
the trisubstituted phenyl piece using cross coupling methodology to
give compound 1. This reaction can typically be carried out using
Suzuki, Stille, Negishi or similar conditions. A typical cross
coupling reaction for the transformation of compound III(B) to
compound I would be to treat compound III(B) with a boronic acid or
ester in the presence of a base such as sodium carbonate and Pd(0)
catalyst in a solvent mixture such as water and 1,4-dioxane to give
compound 1.
[0080] The compounds of the present invention may have asymmetric
carbon atoms. The carbon-carbon bonds of the compounds of the
present invention may be depicted herein using a solid line (--), a
solid wedge (), or a dotted wedge (). The use of a solid line to
depict bonds to asymmetric carbon atoms is meant to indicate that
all possible stereoisomers (e.g. specific enantiomers, racemic
mixtures, etc.) at that carbon atom are included. The use of either
a solid or dotted wedge to depict bonds to asymmetric carbon atoms
is meant to indicate that only the stereoisomer shown is meant to
be included. It is possible that compounds of the invention may
contain more than one asymmetric carbon atom. In those compounds,
the use of a solid line to depict bonds to asymmetric carbon atoms
is meant to indicate that all possible stereoisomers are meant to
be included. For example, unless stated otherwise, it is intended
that the compounds of the present invention can exist as
enantiomers and diastereomers or as racemates and mixtures thereof.
The use of a solid line to depict bonds to one or more asymmetric
carbon atoms in a compound of the invention and the use of a solid
or dotted wedge to depict bonds to other asymmetric carbon atoms in
the same compound is meant to indicate that a mixture of
diastereomers is present.
[0081] Conventional techniques for the preparation/isolation of
individual enantiomers include chiral synthesis from a suitable
optically pure precursor or resolution of the racemate using, for
example, chiral high pressure liquid chromatography (HPLC).
Alternatively, the racemate (or a racemic precursor) may be reacted
with a suitable optically active compound, for example, an alcohol,
or, in the case where the compound contains an acidic or basic
moiety, an acid or base such as tartaric acid or
1-phenylethylamine. The resulting diastereomeric mixture may be
separated by chromatography and/or fractional crystallization and
one or both of the diastereoisomers converted to the corresponding
pure enantiomer(s) by means well known to one skilled in the art.
Chiral compounds of the invention (and chiral precursors thereof)
may be obtained in enantiomerically-enriched form using
chromatography, typically HPLC, on an asymmetric resin with a
mobile phase consisting of a hydrocarbon, typically heptane or
hexane, containing from 0 to 50% isopropanol, typically from 2 to
20%, and from 0 to 5% of an alkylamine, typically 0.1%
diethylamine. Concentration of the eluate affords the enriched
mixture. Stereoisomeric conglomerates may be separated by
conventional techniques known to those skilled in the art. See,
e.g. "Stereochemistry of Organic Compounds" by E. L. Eliel (Wiley,
New York, 1994), the disclosure of which is incorporated herein by
reference in its entirety.
[0082] Where a compound of the invention contains an alkenyl or
alkenylene group, geometric cis/trans (or Z/E) isomers are
possible. Cis/trans isomers may be separated by conventional
techniques well known to those skilled in the art, for example,
chromatography and fractional crystallization. Where structural
isomers are interconvertible via a low energy barrier, tautomeric
isomerism (`tautomerism`) can occur. This can take the form of
proton tautomerism in compounds of the present invention
containing, for example, an imino, keto, or oxime group, or
so-called valence tautomerism in compounds which contain an
aromatic moiety. It follows that a single compound may exhibit more
than one type of isomerism. Included within the scope of the
invention are all stereoisomers, geometric isomers and tautomeric
forms of the inventive compounds, including compounds exhibiting
more than one type of isomerism, and mixtures of one or more
thereof.
[0083] Salts of the present invention can be prepared according to
methods known to those of skill in the art. Examples of salts
include, but are not limited to, acetate, acrylate,
benzenesulfonate, benzoate (such as chlorobenzoate, methylbenzoate,
dinitrobenzoate, hydroxybenzoate, and methoxybenzoate),
bicarbonate, bisulfate, bisulfite, bitartrate, borate, bromide,
butyne-1,4-dioate, calcium edetate, camsylate, carbonate, chloride,
caproate, caprylate, clavulanate, citrate, decanoate,
dihydrochloride, dihydrogenphosphate, edetate, edislyate, estolate,
esylate, ethylsuccinate, formate, fumarate, gluceptate, gluconate,
glutamate, glycollate, glycollylarsanilate, heptanoate,
hexyne-1,6-dioate, hexylresorcinate, hydrabamine, hydrobromide,
hydrochloride, .gamma.-hydroxybutyrate, iodide, isobutyrate,
isothionate, lactate, lactobionate, laurate, malate, maleate,
malonate, mandelate, mesylate, metaphosphate, methane-sulfonate,
methylsulfate, monohydrogenphosphate, mucate, napsylate,
naphthalene-1-sulfonate, naphthalene-2-sulfonate, nitrate, oleate,
oxalate, pamoate (embonate), palmitate, pantothenate,
phenylacetates, phenylbutyrate, phenylpropionate, phthalate,
phospate/diphosphate, polygalacturonate, propanesulfonate,
propionate, propiolate, pyrophosphate, pyrosulfate, salicylate,
stearate, subacetate, suberate, succinate, sulfate, sulfonate,
sulfite, tannate, tartrate, teoclate, tosylate, triethiodode, and
valerate salts.
[0084] The compounds of the present invention that are basic in
nature are capable of forming a wide variety of salts with various
inorganic and organic acids. Although such salts must be
pharmaceutically acceptable for administration to animals, it is
often desirable in practice to initially isolate the compound of
the present invention from the reaction mixture as a
pharmaceutically unacceptable salt and then simply convert the
latter back to the free base compound by treatment with an alkaline
reagent and subsequently convert the latter free base to a
pharmaceutically acceptable acid addition salt. The acid addition
salts of the base compounds of this invention can be prepared by
treating the base compound with a substantially equivalent amount
of the selected mineral or organic acid in an aqueous solvent
medium or in a suitable organic solvent, such as methanol or
ethanol. Upon evaporation of the solvent, the desired solid salt is
obtained. The desired acid salt can also be precipitated from a
solution of the free base in an organic solvent by adding an
appropriate mineral or organic acid to the solution.
[0085] Those compounds of the present invention that are acidic in
nature are capable of forming base salts with various
pharmacologically acceptable cations. Examples of such salts
include the alkali metal or alkaline-earth metal salts and
particularly, the sodium and potassium salts. These salts are all
prepared by conventional techniques. The chemical bases which are
used as reagents to prepare the pharmaceutically acceptable base
salts of this invention are those which form non-toxic base salts
with the acidic compounds of the present invention. Such non-toxic
base salts include those derived from such pharmacologically
acceptable cations as sodium, potassium calcium and magnesium, etc.
These salts may be prepared by any suitable method, for example,
treatment of the free acid with an inorganic or organic base, such
as an amine (primary, secondary or tertiary), an alkali metal
hydroxide or alkaline earth metal hydroxide, or the like.
Illustrative examples of suitable salts include organic salts
derived from amino acids, such as glycine and arginine, ammonia,
primary, secondary, and tertiary amines, and cyclic amines, such as
piperidine, morpholine and piperazine, and inorganic salts derived
from sodium, calcium, potassium, magnesium, manganese, iron,
copper, zinc, aluminum and lithium. These salts can also be
prepared by treating the corresponding acidic compounds with an
aqueous solution containing the desired pharmacologically
acceptable cations, and then evaporating the resulting solution to
dryness, preferably under reduced pressure. Alternatively, they may
also be prepared by mixing lower alkanolic solutions of the acidic
compounds and the desired alkali metal alkoxide together, and then
evaporating the resulting solution to dryness in the same manner as
before. In either case, stoichiometric quantities of reagents are
preferably employed in order to ensure completeness of reaction and
maximum yields of the desired final product.
[0086] If the inventive compound is a base, the desired
pharmaceutically acceptable salt may be prepared by any suitable
method available in the art, for example, treatment of the free
base with an inorganic acid, such as hydrochloric acid, hydrobromic
acid, sulfuric acid, nitric acid, phosphoric acid and the like, or
with an organic acid, such as acetic acid, maleic acid, succinic
acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid,
oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid,
such as glucuronic acid or galacturonic acid, an alpha-hydroxy
acid, such as citric acid or tartaric acid, an amino acid, such as
aspartic acid or glutamic acid, an aromatic acid, such as benzoic
acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic
acid or ethanesulfonic acid, or the like.
[0087] In the case of compounds that are solids, it is understood
by those skilled in the art that the inventive compounds and salts
may exist in different crystalline or polymorphic forms, or in an
amorphous form, all of which are intended to be within the scope of
the present invention.
[0088] The invention also includes isotopically-labeled compounds
of the invention, wherein one or more atoms is replaced by an atom
having the same atomic number, but an atomic mass or mass number
different from the atomic mass or mass number usually found in
nature. Examples of isotopes suitable for inclusion in the
compounds of the invention include isotopes of hydrogen, such as
.sup.2H and .sup.3H, carbon, such as .sup.11C, .sup.13C and
.sup.14C, chlorine, such as .sup.36Cl, fluorine, such as .sup.18F,
iodine, such as .sup.123I and .sup.125I, nitrogen, such as .sup.13N
and .sup.15N, oxygen, such as .sup.15O, .sup.17O and .sup.18O,
phosphorus, such as .sup.32P, and sulfur, such as .sup.35S. Certain
isotopically-labeled compounds of the invention, for example, those
incorporating a radioactive isotope, are useful in drug and/or
substrate tissue distribution studies. The radioactive isotopes
tritium, .sup.3H, and carbon-14, .sup.14C, are particularly useful
for this purpose in view of their ease of incorporation and ready
means of detection. Substitution with heavier isotopes such as
deuterium, 2H, may afford certain therapeutic advantages resulting
from greater metabolic stability, for example, increased in vivo
half-life or reduced dosage requirements, and hence may be
preferred in some circumstances. Substitution with positron
emitting isotopes, such as .sup.11C, .sup.18F, .sup.15O and
.sup.13N, can be useful in Positron Emission Topography (PET)
studies for examining substrate receptor occupancy.
Isotopically-labeled compounds of the invention can generally be
prepared by conventional techniques known to those skilled in the
art or by processes analogous to those described herein, using an
appropriate isotopically-labeled reagent in place of the
non-labeled reagent otherwise employed.
[0089] The compounds of the invention may exist in both unsolvated
and solvated forms. employed when said solvent is water. Examples
of solvate forms include, but are not limited to, compounds of the
invention in association with water, isopropanol, ethanol,
methanol, dimethylsulfoxide (DMSO), ethyl acetate, acetic acid,
ethanolamine, or mixtures thereof. It is specifically contemplated
that in the present invention one solvent molecule can be
associated with one molecule of the compounds of the present
invention, such as a hydrate. dihydrate. Additionally, it is
specifically contemplated that in the present invention less than
one solvent molecule may be associated with one molecule of the
compounds of the present invention, such as a hemihydrate.
Furthermore, solvates of the present invention are contemplated as
solvates of compounds of the present invention that retain the
biological effectiveness of the non-hydrate form of the
compounds.
[0090] Prodrugs of the compounds described herein are also within
the scope of the invention. Thus certain derivatives of the
compounds of the present invention, which derivatives may have
little or no pharmacological activity themselves, when administered
into or onto the body may be converted into compounds of the
present invention having the desired activity, for example, by
hydrolytic cleavage. Such derivatives are referred to as
`prodrugs`. Further information on the use of prodrugs may be found
in Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium
Series (T. Higuchi and W. Stella) and Bioreversible Carriers in
Drug Design, Pergamon Press, 1987 (ed. E. B. Roche, American
Pharmaceutical Association).
[0091] Prodrugs in accordance with the invention can, for example,
be produced by replacing appropriate functionalities present in the
compounds of the present invention with certain moieties known to
those skilled in the art as pro-moieties' as described, for
example, in Design of Prodrugs by H. Bundgaard (Elsevier,
1985).
[0092] Some examples of prodrugs in accordance with the invention
include:
[0093] (i) where the compounds of the present invention contain a
carboxylic acid functionality (--COOH), a prodrug compound wherein
the hydrogen of the carboxylic acid functionality of the compound
is replaced by (C.sub.1-C.sub.8)alkyl to form the corresponding
ester;
[0094] (ii) where the compounds of the present invention contain an
alcohol functionality (--OH), a prodrug compound wherein the
hydrogen of the alcohol functionality of the compound is replaced
by (C.sub.1-C.sub.6) alkanoyloxymethyl to form the corresponding
ether; and
[0095] (iii) where the compounds of the present invention contain a
primary or secondary amino functionality (--NH.sub.2 or --NHR where
R.noteq. H), a prodrug compound wherein, as the case may be, one or
both hydrogens of the amino functionality of the compound I is/are
replaced by (C.sub.1-C.sub.10) alkanoyl to form the corresponding
amide.
[0096] Further examples of replacement groups in accordance with
the foregoing examples and examples of other prodrug types may be
found in the aforementioned references. Moreover, certain compounds
of the present invention may themselves act as prodrugs of other
compounds of the present invention.
[0097] Also included within the scope of the invention are
metabolites of compounds of the present invention, that is,
compounds formed in vivo upon administration of the drug. Some
examples of metabolites in accordance with the invention
include:
[0098] (i) where the compounds of the present invention contain a
methyl group, a hydroxymethyl derivative thereof (e.g.
--CH.sub.3->-CH.sub.2OH);
[0099] (ii) where the compounds of the present invention contain an
alkoxy group, a hydroxy derivative thereof (e.g. --OR
->--OH);
[0100] (iii) where the compounds of the present invention contain a
tertiary amino group, a secondary amino derivative thereof (e.g.
--NR.sup.1R.sup.2->--NHR.sup.1 or --NHR.sup.2);
[0101] (iv) where the compounds of the present invention contain a
secondary amino group, a primary derivative thereof (e.g.
--NHR.sup.1->--NH.sub.2);
[0102] (v) where the compounds of the present invention contain a
phenyl moiety, a phenol derivative thereof (e.g. -Ph->-PhOH);
and
[0103] (vi) where the compounds of the present invention contain an
amide group, a carboxylic acid derivative thereof (e.g.
--CONH.sub.2->COOH).
[0104] Compounds of the invention intended for pharmaceutical use
may be administered as crystalline or amorphous products, or
mixtures thereof. They may be obtained, for example, as solid
plugs, powders, or films by methods such as precipitation,
crystallization, freeze drying, spray drying, or evaporative
drying. Microwave or radio frequency drying may be used for this
purpose.
[0105] The compounds can be administered alone or in combination
with one or more other compounds of the invention, or in
combination with one or more other drugs (or as any combination
thereof). Generally, they will be administered as a formulation in
association with one or more pharmaceutically acceptable
excipients. The term "excipient" is used herein to describe any
ingredient other than the compound(s) of the invention. The choice
of excipient will to a large extent depend on factors such as the
particular mode of administration, the effect of the excipient on
solubility and stability, and the nature of the dosage form.
Pharmaceutical compositions suitable for the delivery of compounds
of the invention and methods for their preparation will be readily
apparent to those skilled in the art. Such compositions and methods
for their preparation can be found, for example, in `Remington's
Pharmaceutical Sciences`, 19th Edition (Mack Publishing Company,
1995), the disclosure of which is incorporated herein by reference
in its entirety.
Oral Administration
[0106] The compounds of the invention may be administered orally.
Oral administration may involve swallowing, so that the compound
enters the gastrointestinal tract, or buccal or sublingual
administration may be employed by which the compound enters the
blood stream directly from the mouth.
[0107] Formulations suitable for oral administration include solid
formulations such as tablets, capsules containing particulates,
liquids, or powders, lozenges (including liquid-filled), chews,
multi- and nano-particulates, gels, solid solution, liposome, films
(including muco-adhesive), ovules, sprays and liquid
formulations.
[0108] Liquid formulations include suspensions, solutions, syrups
and elixirs. Such formulations may be used as fillers in soft or
hard capsules and typically include a carrier, for example, water,
ethanol, polyethylene glycol, propylene glycol, methylcellulose, or
a suitable oil, and one or more emulsifying agents and/or
suspending agents. Liquid formulations may also be prepared by the
reconstitution of a solid, for example, from a sachet.
The compounds of the invention may also be used in fast-dissolving,
fast-disintegrating dosage forms such as those described in Expert
Opinion in Therapeutic Patents, 11 (6), 981-986 by Liang and Chen
(2001), the disclosure of which is incorporated herein by reference
in its entirety. For tablet dosage forms, depending on dose, the
drug may make up from 1 wt % to 80 wt % of the dosage form, more
typically from 5 wt % to 60 wt % of the dosage form. In addition to
the drug, tablets generally contain a disintegrant. Examples of
disintegrants include sodium starch glycolate, sodium carboxymethyl
cellulose, calcium carboxymethyl cellulose, croscarmellose sodium,
crospovidone, polyvinylpyrrolidone, methyl cellulose,
microcrystalline cellulose, lower alkyl-substituted hydroxypropyl
cellulose, starch, pregelatinized starch and sodium alginate.
Generally, the disintegrant will comprise from 1 wt % to 25 wt %,
preferably from 5 wt % to 20 wt % of the dosage form.
[0109] Binders are generally used to impart cohesive qualities to a
tablet formulation. Suitable binders include microcrystalline
cellulose, gelatin, sugars, polyethylene glycol, natural and
synthetic gums, polyvinylpyrrolidone, pregelatinized starch,
hydroxypropyl cellulose and hydroxypropyl methylcellulose. Tablets
may also contain diluents, such as lactose (monohydrate,
spray-dried monohydrate, anhydrous and the like), mannitol,
xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose,
starch and dibasic calcium phosphate dihydrate.
Tablets may also optionally include surface active agents, such as
sodium lauryl sulfate and polysorbate 80, and glidants such as
silicon dioxide and talc. When present, surface active agents are
typically in amounts of from 0.2 wt % to 5 wt % of the tablet, and
glidants typically from 0.2 wt % to 1 wt % of the tablet.
[0110] Tablets also generally contain lubricants such as magnesium
stearate, calcium stearate, zinc stearate, sodium stearyl fumarate,
and mixtures of magnesium stearate with sodium lauryl sulphate.
Lubricants generally are present in amounts from 0.25 wt % to 10 wt
%, preferably from 0.5 wt % to 3 wt % of the tablet.
Other conventional ingredients include anti-oxidants, colorants,
flavoring agents, preservatives and taste-masking agents.
[0111] Exemplary tablets contain up to about 80 wt % drug, from
about 10 wt % to about 90 wt % binder, from about 0 wt % to about
85 wt % diluent, from about 2 wt % to about 10 wt % disintegrant,
and from about 0.25 wt % to about 10 wt % lubricant.
Tablet blends may be compressed directly or by roller to form
tablets. Tablet blends or portions of blends may alternatively be
wet-, dry-, or melt-granulated, melt congealed, or extruded before
tabletting. The final formulation may include one or more layers
and may be coated or uncoated; or encapsulated.
[0112] The formulation of tablets is discussed in detail in
"Pharmaceutical Dosage Forms Tablets, Vol. 1", by H. Lieberman and
L. Lachman, Marcel Dekker, N.Y., N.Y., 1980 (ISBN 0-8247-6918-X),
the disclosure of which is incorporated herein by reference in its
entirety.
[0113] Solid formulations for oral administration may be formulated
to be immediate and/or modified release. Modified release
formulations include delayed-, sustained-, pulsed-, controlled-,
targeted and programmed release.
[0114] Suitable modified release formulations are described in U.S.
Pat. No. 6,106,864. Details of other suitable release technologies
such as high energy dispersions and osmotic and coated particles
can be found in Verma et al, Pharmaceutical Technology On-line,
25(2), 1-14 (2001). The use of chewing gum to achieve controlled
release is described in WO 00/35298. The disclosures of these
references are incorporated herein by reference in their
entireties.
Parenteral Administration
[0115] The compounds of the invention may also be administered
directly into the blood stream, into muscle, or into an internal
organ. Suitable means for parenteral administration include
intravenous, intraarterial, intraperitoneal, intrathecal,
intraventricular, intraurethral, intrasternal, intracranial,
intramuscular and subcutaneous. Suitable devices for parenteral
administration include needle (including microneedle) injectors,
needle-free injectors and infusion techniques.
[0116] Parenteral formulations are typically aqueous solutions
which may contain excipients such as salts, carbohydrates and
buffering agents (preferably to a pH of from 3 to 9), but, for some
applications, they may be more suitably formulated as a sterile
non-aqueous solution or as a dried form to be used in conjunction
with a suitable vehicle such as sterile, pyrogen-free water.
[0117] The preparation of parenteral formulations under sterile
conditions, for example, by lyophilization, may readily be
accomplished using standard pharmaceutical techniques well known to
those skilled in the art. The solubility of compounds of the
invention used in the preparation of parenteral solutions may be
increased by the use of appropriate formulation techniques, such as
the incorporation of solubility-enhancing agents.
[0118] Formulations for parenteral administration may be formulated
to be immediate and/or modified release. Modified release
formulations include delayed-, sustained-, pulsed-, controlled-,
targeted and programmed release. Thus compounds of the invention
may be formulated as a solid, semi-solid, or thixotropic liquid for
administration as an implanted depot providing modified release of
the active compound. Examples of such formulations include
drug-coated stents and PGLA microspheres.
Topical Administration
[0119] The compounds of the invention may also be administered
topically to the skin or mucosa, that is, dermally or
transdermally. Typical formulations for this purpose include gels,
hydrogels, lotions, solutions, creams, ointments, dusting powders,
dressings, foams, films, skin patches, wafers, implants, sponges,
fibers, bandages and microemulsions. Liposomes may also be used.
Typical carriers include alcohol, water, mineral oil, liquid
petrolatum, white petrolatum, glycerin, polyethylene glycol and
propylene glycol. Penetration enhancers may be incorporated; see,
for example, J Pharm Sci, 88 (10), 955-958 by Finnin and Morgan
(October 1999). Other means of topical administration include
delivery by electroporation, iontophoresis, phonophoresis,
sonophoresis and microneedle or needle-free (e.g. Powderject.TM.,
Bioject.TM., etc.) injection. The disclosures of these references
are incorporated herein by reference in their entireties.
[0120] Formulations for topical administration may be formulated to
be immediate and/or modified release. Modified release formulations
include delayed-, sustained-, pulsed-, controlled-, targeted and
programmed release.
Inhaled/Intranasal Administration
[0121] The compounds of the invention can also be administered
intranasally or by inhalation, typically in the form of a dry
powder (either alone, as a mixture, for example, in a dry blend
with lactose, or as a mixed component particle, for example, mixed
with phospholipids, such as phosphatidylcholine) from a dry powder
inhaler or as an aerosol spray from a pressurized container, pump,
spray, atomizer (preferably an atomizer using electrohydrodynamics
to produce a fine mist), or nebulizer, with or without the use of a
suitable propellant, such as 1,1,1,2-tetrafluoroethane or
1,1,1,2,3,3,3-heptafluoropropane. For intranasal use, the powder
may include a bioadhesive agent, for example, chitosan or
cyclodextrin.
[0122] The pressurized container, pump, spray, atomizer, or
nebulizer contains a solution or suspension of the compound(s) of
the invention comprising, for example, ethanol, aqueous ethanol, or
a suitable alternative agent for dispersing, solubilizing, or
extending release of the active, a propellant(s) as solvent and an
optional surfactant, such as sorbitan trioleate, oleic acid, or an
oligolactic acid.
[0123] Prior to use in a dry powder or suspension formulation, the
drug product is micronized to a size suitable for delivery by
inhalation (typically less than 5 microns). This may be achieved by
any appropriate comminuting method, such as spiral jet milling,
fluid bed jet milling, supercritical fluid processing to form
nanoparticles, high pressure homogenisation, or spray drying.
[0124] Capsules (made, for example, from gelatin or HPMC), blisters
and cartridges for use in an inhaler or insufflator may be
formulated to contain a powder mix of the compound of the
invention, a suitable powder base such as lactose or starch and a
performance modifier such as I-leucine, mannitol, or magnesium
stearate. The lactose may be anhydrous or in the form of the
monohydrate, preferably the latter. Other suitable excipients
include dextran, glucose, maltose, sorbitol, xylitol, fructose,
sucrose and trehalose.
[0125] A suitable solution formulation for use in an atomizer using
electrohydrodynamics to produce a fine mist may contain from 1
.mu.g to 20 mg of the compound of the invention per actuation and
the actuation volume may vary from 1 .mu.L to 100 .mu.L. A typical
formulation includes a compound of the invention, propylene glycol,
sterile water, ethanol and sodium chloride. Alternative solvents
which may be used instead of propylene glycol include glycerol and
polyethylene glycol.
[0126] Suitable flavors, such as menthol and levomenthol, or
sweeteners, such as saccharin or saccharin sodium, may be added to
those formulations of the invention intended for inhaled/intranasal
administration.
[0127] Formulations for inhaled/intranasal administration may be
formulated to be immediate and/or modified release using, for
example, poly(DL-lactic-coglycolic acid (PGLA). Modified release
formulations include delayed-, sustained-, pulsed-, controlled-,
targeted and programmed release.
[0128] In the case of dry powder inhalers and aerosols, the dosage
unit is determined by means of a valve which delivers a metered
amount. Units in accordance with the invention are typically
arranged to administer a metered dose or "puff" containing a
desired mount of the compound of the invention. The overall daily
dose may be administered in a single dose or, more usually, as
divided doses throughout the day.
Rectal/Intravaginal Administration
[0129] Compounds of the invention may be administered rectally or
vaginally, for example, in the form of a suppository, pessary, or
enema. Cocoa butter is a traditional suppository base, but various
alternatives may be used as appropriate.
[0130] Formulations for rectal/vaginal administration may be
formulated to be immediate and/or modified release. Modified
release formulations include delayed-, sustained-, pulsed-,
controlled-, targeted and programmed release.
Ocular Administration
[0131] Compounds of the invention may also be administered directly
to the eye or ear, typically in the form of drops of a micronized
suspension or solution in isotonic, pH-adjusted, sterile saline.
Other formulations suitable for ocular and aural administration
include ointments, biodegradable (e.g. absorbable gel sponges,
collagen) and non-biodegradable (e.g. silicone) implants, wafers,
lenses and particulate or vesicular systems, such as niosomes or
liposomes. A polymer such as crossed-linked polyacrylic acid,
polyvinylalcohol, hyaluronic acid, a cellulosic polymer, for
example, hydroxypropylmethylcellulose, hydroxyethylcellulose, or
methyl cellulose, or a heteropolysaccharide polymer, for example,
gelan gum, may be incorporated together with a preservative, such
as benzalkonium chloride. Such formulations may also be delivered
by iontophoresis.
[0132] Formulations for ocular/aural administration may be
formulated to be immediate and/or modified release. Modified
release formulations include delayed-, sustained-, pulsed-,
controlled-, targeted, or programmed release.
Other Technologies
[0133] Compounds of the invention may be combined with soluble
macromolecular entities, such as cyclodextrin and suitable
derivatives thereof or polyethylene glycol-containing polymers, in
order to improve their solubility, dissolution rate, taste-masking,
bioavailability and/or stability for use in any of the
aforementioned modes of administration.
[0134] Drug-cyclodextrin complexes, for example, are found to be
generally useful for most dosage forms and administration routes.
Both inclusion and non-inclusion complexes may be used. As an
alternative to direct complexation with the drug, the cyclodextrin
may be used as an auxiliary additive, i.e. as a carrier, diluent,
or solubilizer. Most commonly used for these purposes are alpha-,
beta- and gamma-cyclodextrins, examples of which may be found in
PCT Publication Nos. WO 91/11172, WO 94/02518 and WO 98/55148, the
disclosures of which are incorporated herein by reference in their
entireties.
[0135] The amount of the active compound administered will be
dependent on the subject being treated, the severity of the
disorder or condition, the rate of administration, the disposition
of the compound and the discretion of the prescribing physician.
However, an effective dosage is typically in the range of about
0.001 to about 100 mg per kg body weight per day, preferably about
0.01 to about 35 mg/kg/day, in single or divided doses. For a 70 kg
human, this would amount to about 0.07 to about 7000 mg/day,
preferably about 0.7 to about 2500 mg/day. In some instances,
dosage levels below the lower limit of the aforesaid range may be
more than adequate, while in other cases still larger doses may be
used without causing any harmful side effect, with such larger
doses typically divided into several smaller doses for
administration throughout the day.
[0136] This invention also relates to a method for the treatment of
abnormal cell growth in a mammal which comprises administering to
said mammal an amount of a compound of the present invention, or a
salt or solvate thereof, that is effective in treating abnormal
cell growth in combination with an anti-tumor agent selected from
the group consisting of mitotic inhibitors, alkylating agents,
anti-metabolites, intercalating antibiotics, growth factor
inhibitors, cell cycle inhibitors, enzymes, topoisomerase
inhibitors, biological response modifiers, antibodies, cytotoxics,
anti-hormones, and anti-androgens.
[0137] In one embodiment of the present invention the anti-tumor
agent used in conjunction with a compound of the present invention
and pharmaceutical compositions described herein is an
anti-angiogenesis agent, kinase inhibitor, pan kinase inhibitor or
growth factor inhibitor. Preferred pan kinase inhibitors include
Sutent.TM. (sunitinib), described in U.S. Pat. No. 6,573,293
(Pfizer, Inc, NY, USA). Anti-angiogenesis agents, include but are
not limited to the following agents, such as EGF inhibitors, EGFR
inhibitors, VEGF inhibitors, VEGFR inhibitors, TIE2 inhibitors,
IGF1R inhibitors, COX-II (cyclooxygenase II) inhibitors, MMP-2
(matrix-metalloproteinase 2) inhibitors, and MMP-9
(matrix-metalloproteinase 9) inhibitors.
[0138] Preferred VEGF inhibitors, include for example, Avastin
(bevacizumab), an anti-VEGF monoclonal antibody of Genentech, Inc.
of South San Francisco, Calif. Additional VEGF inhibitors include
CP-547,632 (Pfizer Inc., NY, USA), AG13736 (Pfizer Inc.), ZD-6474
(AstraZeneca), AEE788 (Novartis), AZD-2171, VEGF Trap
(Regeneron/Aventis), Vatalanib (also known as PTK-787, ZK-222584:
Novartis & Schering AG), Macugen (pegaptanib octasodium,
NX-1838, EYE-001, Pfizer Inc./Gilead/Eyetech), IM862 (Cytran Inc.
of Kirkland, Wash., USA); and angiozyme, a synthetic ribozyme from
Ribozyme (Boulder, Colo.) and Chiron (Emeryville, Calif.) and
combinations thereof.
[0139] VEGF inhibitors useful in the practice of the present
invention are described in U.S. Pat. Nos. 6,534,524 and 6,235,764,
both of which are incorporated in their entirety for all purposes.
Additional VEGF inhibitors are described in, for example in WO
99/24440, in WO 95/21613, WO 99/61422, U.S. Pat. No. 5,834,504, WO
98/50356, U.S. Pat. No. 5,883,113 U.S. Pat. No. 5,886,020, U.S.
Pat. No. 5,792,783, U.S. Pat. No. 6,653,308, WO 99/10349, WO
97/32856, WO 97/22596, WO 98/54093, WO 98/02438, WO 99/16755, and
WO 98/02437, all of which are herein incorporated by reference in
their entirety.
[0140] Other anti-angiogenic compounds include acitretin,
fenretinide, thalidomide, zoledronic acid, angiostatin, aplidine,
cilengtide, combretastatin A-4, endostatin, halofuginone,
rebimastat, removab, Revlimid, squalamine, ukrain, Vitaxin and
combinations thereof.
Other antiproliferative agents that may be used in combination with
the compounds of the present invention include inhibitors of the
enzyme farnesyl protein transferase and inhibitors of the receptor
tyrosine kinase PDGFr, including the compounds disclosed and
claimed in the following: U.S. Pat. No. 6,080,769; U.S. Pat. No.
6,194,438; U.S. Pat. No. 6,258,824; U.S. Pat. No. 6,586447; U.S.
Pat. No. 6,071,935; U.S. Pat. No. 6,495,564; and U.S. Pat. No.
6,150,377; U.S. Pat. No. 6,596,735; U.S. Pat. No. 6,479,513; WO
01/40217; U.S. 2003-0166675. Each of the foregoing patents and
patent applications is herein incorporated by reference in their
entirety.
[0141] PDGRr inhibitors include but are not limited to those
disclosed in international patent application publication numbers
WO01/40217 and WO2004/020431, the contents of which are
incorporated in their entirety for all purposes. Preferred PDGFr
inhibitors include Pfizer's CP-673,451 and CP-868,596 and its
salts.
[0142] Preferred GARF inhibitors include Pfizer's AG-2037
(pelitrexol and its salts). GARF inhibitors useful in the practice
of the present invention are disclosed in U.S. Pat. No. 5,608,082
which is incorporated in its entirety for all purposes.
[0143] Examples of useful COX-II inhibitors which can be used in
conjunction with a compound of Formula (I) and pharmaceutical
compositions disclosed herein include CELEBREX.TM. (celecoxib),
parecoxib, deracoxib, ABT-963, MK-663 (etoricoxib), COX-189
(Lumiracoxib), BMS 347070, RS 57067, NS-398, Bextra (valdecoxib),
paracoxib, Vioxx (rofecoxib), SD-8381,
4-Methyl-2-(3,4-dimethylphenyl)-1-(4-sulfamoyl-phenyl)-1H-pyrrole,
2-(4-Ethoxyphenyl)-4-methyl-1-(4-sulfamoylphenyl)-1H-pyrrole,
T-614, JTE-522, S-2474, SVT-2016, CT-3, SC-58125 and Arcoxia
(etoricoxib). Additionally, COX-II inhibitors are disclosed in U.S.
Patent Applications US 2005-0148627 and US 2005-0148777, the
contents of which are incorporated in their entirety for all
purposes.
[0144] In a particular embodiment the anti-tumor agent is celecoxib
(U.S. Pat. No. 5,466,823), valdecoxib (U.S. Pat. No. 5,633,272),
parecoxib (U.S. Pat. No. 5,932,598), deracoxib (U.S. Pat. No.
5,521,207), SD-8381 (U.S. Pat. No. 6,034,256, Example 175), ABT-963
(WO 2002/24719), rofecoxib (CAS No. 162011-90-7), MK-663 (or
etoricoxib) as disclosed in WO 1998/03484, COX-189 (Lumiracoxib) as
disclosed in WO 1999/11605, BMS-347070 (U.S. Pat. No. 6,180,651),
NS-398 (CAS 123653-11-2), RS 57067 (CAS 17932-91-3),
4-Methyl-2-(3,4-dimethylphenyl)-1-(4-sulfamoyl-phenyl)-1H-pyrrole,
2-(4-Ethoxyphenyl)-4-methyl-1-(4-sulfamoylphenyl)-1H-pyrrole, or
meloxicam.
[0145] Other useful inhibitors as anti-tumor agents used in
combination with a compound of the present invention and
pharmaceutical compositions disclosed herein include aspirin, and
non-steroidal anti-inflammatory drugs (NSAIDs) which inhibit the
enzyme that makes prostaglandins (cyclooxygenase I and II),
resulting in lower levels of prostaglandins, include but are not
limited to the following, Salsalate (Amigesic), Diflunisal
(Dolobid), Ibuprofen (Motrin), Ketoprofen (Orudis), Nabumetone
(Relafen), Piroxicam (Feldene), Naproxen (Aleve, Naprosyn),
Diclofenac (Voltaren), Indomethacin (Indocin), Sulindac (Clinoril),
Tolmetin (Tolectin), Etodolac (Lodine), Ketorolac (Toradol),
Oxaprozin (Daypro) and combinations thereof.
[0146] Preferred COX-I inhibitors include ibuprofen (Motrin),
nuprin, naproxen (Aleve), indomethacin (Indocin), nabumetone
(Relafen) and combinations thereof.
[0147] Targeted agents used in combination with a compound of the
present invention and pharmaceutical compositions disclosed herein
include EGFr inhibitors such as Iressa (gefitinib, AstraZeneca),
Tarceva (erlotinib or OSI-774, OSI Pharmaceuticals Inc.), Erbitux
(cetuximab, Imclone Pharmaceuticals, Inc.), EMD-7200 (Merck AG),
ABX-EGF (Amgen Inc. and Abgenix Inc.), HR3 (Cuban Government), IgA
antibodies (University of Erlangen-Nuremberg), TP-38 (IVAX), EGFR
fusion protein, EGF-vaccine, anti-EGFr immunoliposomes (Hermes
Biosciences Inc.) and combinations thereof. Preferred EGFr
inhibitors include Iressa, Erbitux, Tarceva and combinations
thereof.
[0148] Other anti-tumor agents include those selected from pan erb
receptor inhibitors or ErbB2 receptor inhibitors, such as
CP-724,714 (Pfizer, Inc.), Cl-1033 (canertinib, Pfizer, Inc.),
Herceptin (trastuzumab, Genentech Inc.), Omitarg (2C4, pertuzumab,
Genentech Inc.), TAK-165 (Takeda), GW-572016 (Ionafarnib,
GlaxoSmithKline), GW-282974 (GlaxoSmithKline), EKB-569 (Wyeth),
PKI-166 (Novartis), dHER2 (HER2 Vaccine, Corixa and
GlaxoSmithKline), APC8024 (HER2 Vaccine, Dendreon), anti-HER2/neu
bispecific antibody (Decof Cancer Center), B7.her2.IgG3 (Agensys),
AS HER2 (Research Institute for Rad Biology & Medicine),
trifunctional bispecific antibodies (University of Munich) and mAB
AR-209 (Aronex Pharmaceuticals Inc) and mAB 2B-1 (Chiron) and
combinations thereof.
[0149] Preferred erb selective anti-tumor agents include Herceptin,
TAK-165, CP-724,714, ABX-EGF, HER3 and combinations thereof.
Preferred pan erbb receptor inhibitors include GW572016, Cl-1033,
EKB-569, and Omitarg and combinations thereof.
Additional erbB2 inhibitors include those disclosed in WO 98/02434,
WO 99/35146, WO 99/35132, WO 98/02437, WO 97/13760, WO 95/19970,
U.S. Pat. No. 5,587,458, and U.S. Pat. No. 5,877,305, each of which
is herein incorporated by reference in its entirety. ErbB2 receptor
inhibitors useful in the present invention are also disclosed in
U.S. Pat. Nos. 6,465,449, and 6,284,764, and in WO 2001/98277 each
of which are herein incorporated by reference in their
entirety.
[0150] Additionally, other anti-tumor agents may be selected from
the following agents, BAY-43-9006 (Onyx Pharmaceuticals Inc.),
Genasense (augmerosen, Genta), Panitumumab (Abgenix/Amgen), Zevalin
(Schering), Bexxar (Corixa/GlaxoSmithKline), Abarelix, Alimta, EPO
906 (Novartis), discodermolide (XAA-296), ABT-510 (Abbott),
Neovastat (Aeterna), enzastaurin (Eli Lilly), Combrestatin A4P
(Oxigene), ZD-6126 (AstraZeneca), flavopiridol (Aventis), CYC-202
(Cyclacel), AVE-8062 (Aventis), DMXAA (Roche/Antisoma), Thymitaq
(Eximias), Temodar (temozolomide, Schering Plough) and Revilimd
(Celegene) and combinations thereof.
[0151] Other anti-tumor agents may be selected from the following
agents, CyPat (cyproterone acetate), Histerelin (histrelin
acetate), Plenaixis (abarelix depot), Atrasentan (ABT-627),
Satraplatin (JM-216), thalomid (Thalidomide), Theratope, Temilifene
(DPPE), ABI-007 (paclitaxel), Evista (raloxifene), Atamestane
(Biomed-777), Xyotax (polyglutamate paclitaxel), Targetin
(bexarotine) and combinations thereof.
[0152] Additionally, other anti-tumor agents may be selected from
the following agents, Trizaone (tirapazamine), Aposyn (exisulind),
Nevastat (AE-941), Ceplene (histamine dihydrochloride), Orathecin
(rubitecan), Virulizin, Gastrimmune (G17DT), DX-8951f (exatecan
mesylate), Onconase (ranpirnase), BEC2 (mitumoab), Xcytrin
(motexafin gadolinium) and combinations thereof. Further anti-tumor
agents may be selected from the following agents, CeaVac (CEA),
NeuTrexin (trimetresate glucuronate) and combinations thereof.
Additional anti-tumor agents may be selected from the following
agents, OvaRex (oregovomab), Osidem (IDM-1), and combinations
thereof. Additional anti-tumor agents may be selected from the
following agents, Advexin (ING 201), Tirazone (tirapazamine), and
combinations thereof. Additional anti-tumor agents may be selected
from the following agents, RSR13 (efaproxiral), Cotara (131I chTNT
1/b), NBI-3001 (IL-4) and combinations thereof. Additional
anti-tumor agents may be selected from the following agents,
Canvaxin, GMK vaccine, PEG Interon A, Taxoprexin (DHA/paciltaxel),
and combinations thereof.
Other anti-tumor agents include Pfizer's MEK1/2 inhibitor PD325901,
Array Biopharm's MEK inhibitor ARRY-142886, Bristol Myers' CDK2
inhibitor BMS-387,032, Pfizer's CDK inhibitor PD0332991 and
AstraZeneca's AXD-5438, and combinations thereof.
[0153] Additionally, mTOR inhibitors may also be utilized such as
CCl-779 (Wyeth) and rapamycin derivatives RAD001 (Novartis) and
AP-23573 (Ariad), HDAC inhibitors, SAHA (Merck Inc./Aton
Pharmaceuticals) and combinations thereof. Additional anti-tumor
agents include aurora 2 inhibitor VX-680 (Vertex), and Chk1/2
inhibitor XL844 (Exilixis).
[0154] The following cytotoxic agents, e.g., one or more selected
from the group consisting of epirubicin (Ellence), docetaxel
(Taxotere), paclitaxel, Zinecard (dexrazoxane), rituximab (Rituxan)
imatinib mesylate (Gleevec), and combinations thereof, may be used
in combination with a compound of the present invention and
pharmaceutical compositions disclosed herein.
The invention also contemplates the use of the compounds of the
present invention together with hormonal therapy, including but not
limited to, exemestane (Aromasin, Pfizer Inc.), leuprorelin (Lupron
or Leuplin, TAP/Abbott/Takeda), anastrozole (Arimidex,
Astrazeneca), gosrelin (Zoladex, AstraZeneca), doxercalciferol,
fadrozole, formestane, tamoxifen citrate (tamoxifen, Nolvadex,
AstraZeneca), Casodex (AstraZeneca), Abarelix (Praecis), Trelstar,
and combinations thereof.
[0155] The invention also relates to the use of the compounds of
the present invention together with hormonal therapy agents such as
anti-estrogens including, but not limited to fulvestrant,
toremifene, raloxifene, lasofoxifene, letrozole (Femara, Novartis),
anti-androgens such as bicalutamide, flutamide, mifepristone,
nilutamide, Casodex.TM.
(4'-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3'-(trifluoromet-
hyl)propionanilide, bicalutamide) and combinations thereof.
[0156] Further, the invention provides a compound of the present
invention alone or in combination with one or more supportive care
products, e.g., a product selected from the group consisting of
Filgrastim (Neupogen), ondansetron (Zofran), Fragmin, Procrit,
Aloxi, Emend, or combinations thereof.
[0157] Particularly preferred cytotoxic agents include Camptosar,
Erbitux, Iressa, Gleevec, Taxotere and combinations thereof.
[0158] The following topoisomerase I inhibitors may be utilized as
anti-tumor agents: camptothecin; irinotecan HCl (Camptosar);
edotecarin; orathecin (Supergen); exatecan (Daiichi); BN-80915
(Roche); and combinations thereof. Particularly preferred
toposimerase II inhibitors include epirubicin (Ellence).
[0159] Alkylating agents include, but are not limited to, nitrogen
mustard N-oxide, cyclophosphamide, ifosfamide, melphalan, busulfan,
mitobronitol, carboquone, thiotepa, ranimustine, nimustine,
temozolomide, AMD-473, altretamine, AP-5280, apaziquone,
brostallicin, bendamustine, carmustine, estramustine, fotemustine,
glufosfamide, ifosfamide, KW-2170, mafosfamide, and mitolactol;
platinum-coordinated alkylating compounds include but are not
limited to, cisplatin, Paraplatin (carboplatin), eptaplatin,
lobaplatin, nedaplatin, Eloxatin (oxaliplatin, Sanofi) or
satrplatin and combinations thereof. Particularly preferred
alkylating agents include Eloxatin (oxaliplatin).
[0160] Antimetabolites include but are not limited to,
methotrexate, 6-mercaptopurine riboside, mercaptopurine,
5-fluorouracil (5-FU) alone or in combination with leucovorin,
tegafur, UFT, doxifluridine, carmofur, cytarabine, cytarabine
ocfosfate, enocitabine, S-1, Alimta (premetrexed disodium,
LY231514, MTA), Gemzar (gemcitabine, Eli Lilly), fludarabin,
5-azacitidine, capecitabine, cladribine, clofarabine, decitabine,
eflornithine, ethynylcytidine, cytosine arabinoside, hydroxyurea,
TS-1, melphalan, nelarabine, nolatrexed, ocfosfate, disodium
premetrexed, pentostatin, pelitrexol, raltitrexed, triapine,
trimetrexate, vidarabine, vincristine, vinorelbine; or for example,
one of the preferred anti-metabolites disclosed in European Patent
Application No. 239362 such as
N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamin-
o]-2-thenoyl)-L-glutamic acid and combinations thereof.
Antibiotics include intercalating antibiotics and include, but are
not limited to: aclarubicin, actinomycin D, amrubicin, annamycin,
adriamycin, bleomycin, daunorubicin, doxorubicin, elsamitrucin,
epirubicin, galarubicin, idarubicin, mitomycin C, nemorubicin,
neocarzinostatin, peplomycin, pirarubicin, rebeccamycin,
stimalamer, streptozocin, valrubicin, zinostatin and combinations
thereof.
[0161] Plant derived anti-tumor substances include for example
those selected from mitotic inhibitors, for example vinblastine,
docetaxel (Taxotere), paclitaxel and combinations thereof.
[0162] Cytotoxic topoisomerase inhibiting agents include one or
more agents selected from the group consisting of aclarubicn,
amonafide, belotecan, camptothecin, 10-hydroxycamptothecin,
9-aminocamptothecin, diflomotecan, irinotecan HCl (Camptosar),
edotecarin, epirubicin (Ellence), etoposide, exatecan, gimatecan,
lurtotecan, mitoxantrone, pirarubicin, pixantrone, rubitecan,
sobuzoxane, SN-38, tafluposide, topotecan, and combinations
thereof.
[0163] Preferred cytotoxic topoisomerase inhibiting agents include
one or more agents selected from the group consisting of
camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin,
irinotecan HCl (Camptosar), edotecarin, epirubicin (Ellence),
etoposide, SN-38, topotecan, and combinations thereof.
[0164] Immunologicals include interferons and numerous other immune
enhancing agents. Interferons include interferon alpha, interferon
alpha-2a, interferon, alpha-2b, interferon beta, interferon
gamma-1a, interferon gamma-1b (Actimmune), or interferon gamma-n1
and combinations thereof. Other agents include filgrastim,
lentinan, sizofilan, TheraCys, ubenimex, WF-10, aldesleukin,
alemtuzumab, BAM-002, dacarbazine, daclizumab, denileukin,
gemtuzumab ozogamicin, ibritumomab, imiquimod, lenograstim,
lentinan, melanoma vaccine (Corixa), molgramostim, OncoVAX-CL,
sargramostim, tasonermin, tecleukin, thymalasin, tositumomab,
Virulizin, Z-100, epratuzumab, mitumomab, oregovomab, pemtumomab
(Y-muHMFG1), Provenge (Dendreon) and combinations thereof.
[0165] Biological response modifiers are agents that modify defense
mechanisms of living organisms or biological responses, such as
survival, growth, or differentiation of tissue cells to direct them
to have anti-tumor activity. Such agents include krestin, lentinan,
sizofuran, picibanil, ubenimex and combinations thereof.
[0166] Other anticancer agents that can be used in combination with
a compound of the present invention include alitretinoin, ampligen,
atrasentan bexarotene, bortezomib. Bosentan, calcitriol, exisulind,
finasteride, fotemustine, ibandronic acid, miltefosine,
mitoxantrone, 1-asparaginase, procarbazine, dacarbazine,
hydroxycarbamide, pegaspargase, pentostatin, tazarotne, Telcyta
(TLK-286, Telik Inc.), Velcade (bortemazib, Millenium), tretinoin,
and combinations thereof.
[0167] Platinum-coordinated compounds include but are not limited
to, cisplatin, carboplatin, nedaplatin, oxaliplatin, and
combinations thereof.
[0168] Camptothecin derivatives include but are not limited to
camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin,
irinotecan, SN-38, edotecarin, topotecan and combinations
thereof.
[0169] Other antitumor agents include mitoxantrone, 1-asparaginase,
procarbazine, dacarbazine, hydroxycarbamide, pentostatin, tretinoin
and combinations thereof.
[0170] Anti-tumor agents capable of enhancing antitumor immune
responses, such as CTLA4 (cytotoxic lymphocyte antigen 4)
antibodies, and other agents capable of blocking CTLA4 may also be
utilized, such as MDX-010 (Medarex) and CTLA4 compounds disclosed
in U.S. Pat. No. 6,682,736; and anti-proliferative agents such as
other farnesyl protein transferase inhibitors, for example the
farnesyl protein transferase inhibitors. Additionally, specific
CTLA4 antibodies that can be used in combination with compounds of
the present invention include those disclosed in U.S. Pat. Nos.
6,682,736 and 6,682,736 both of which are herein incorporated by
reference in their entirety.
[0171] Specific IGF1R antibodies that can be used in the
combination methods of the present invention include those
disclosed in WO 2002/053596, which is herein incorporated by
reference in its entirety.
[0172] Specific CD40 antibodies that can be used in the present
invention include those disclosed in WO 2003/040170 which is herein
incorporated by reference in its entirety.
Gene therapy agents may also be employed as anti-tumor agents such
as TNFerade (GeneVec), which express TNFalpha in response to
radiotherapy.
[0173] In one embodiment of the present invention statins may be
used in combination with a compound of the present invention and
pharmaceutical compositions thereof. Statins (HMG-COA reducatase
inhibitors) may be selected from the group consisting of
Atorvastatin (Lipitor.TM., Pfizer Inc.), Provastatin
(Pravachol.TM., Bristol-Myers Squibb), Lovastatin (Mevacor.TM.,
Merck Inc.), Simvastatin (Zocor.TM., Merck Inc.), Fluvastatin
(LeScol.TM., Novartis), Cerivastatin (Baycol.TM., Bayer),
Rosuvastatin (Crestor.TM., AstraZeneca), Lovostatin and Niacin
(Advicor.TM., Kos Pharmaceuticals), derivatives and combinations
thereof.
[0174] In a preferred embodiment the statin is selected from the
group consisting of Atovorstatin and Lovastatin, derivatives and
combinations thereof. Other agents useful as anti-tumor agents
include Caduet.
[0175] Inasmuch as it may desirable to administer a combination of
active compounds, for example, for the purpose of treating a
particular disease or condition, it is within the scope of the
present invention that two or more pharmaceutical compositions, at
least one of which contains a compound in accordance with the
invention, may conveniently be combined in the form of a kit
suitable for coadministration of the compositions. Thus the kit of
the invention includes two or more separate pharmaceutical
compositions, at least one of which contains a compound of the
invention, and means for separately retaining said compositions,
such as a container, divided bottle, or divided foil packet. An
example of such a kit is the familiar blister pack used for the
packaging of tablets, capsules and the like.
[0176] The kit of the invention is particularly suitable for
administering different dosage forms, for example, oral and
parenteral, for administering the separate compositions at
different dosage intervals, or for titrating the separate
compositions against one another. To assist compliance, the kit
typically includes directions for administration and may be
provided with a memory aid.
EXAMPLES
[0177] In the following examples molecules with a single chiral
center, unless otherwise noted or indicated by the structural
formula or chemical name, exist as a racemic mixture. Those
molecules with two or more chiral centers, unless otherwise noted
or indicated by the structural formula or chemical name, exist as a
racemic mixture of diastereomers. Single enantiomers/diastereomers
may be obtained by methods known to those skilled in the art.
[0178] .sup.1H-NMR spectra were recorded on a Bruker instrument
operating either at 300 MHz, or 400 MHz and .sup.13C-NMR spectra
were recorded operating at 75 MHz.
[0179] The following abbreviations may be used herein: Et.sub.2O
(diethyl ether); DMF (N,N-dimethylformamide); THF
(tetrahydrofuran); DCM (dichloro-methane); DMA (dimethyl acetal);
DBU (1,8-diazabicyclo[5.4.0]undec-7-ene); LiHMDS or LHMDS (lithium
hexamethyldisilazide); TBME (tert-butyl methyl ether); LDA (lithium
diisopropylamide); DMSO or dmso (dimethylsulfoxide); MeOH
(methanol); EtOH (ethanol); BuOH (butanol); EtOAc (ethyl acetate);
THF (tetrahydrofuran); Ac (acetyl); Me (methyl); Et (ethyl); Ph
(phenyl); TMSI (trimethylsilyliodide); DSC(N,N'-disuccinimidyl
carbonate); CDI (1,1'-carbonyldiimidazole); Boc
(tert-butoxycarbonyl); nBuLi (n-butyl lithium); EDC
(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride); HOBt
(N-hydroxybenzitriazole hydrate); DME (1,2-dimethoxyethane);
Pd(dba).sub.2 (bis(dibenzylideneacetone)palladium(0)); and RT or rt
(room temperature).
Example 1
2-amino-N-bicyclo[1.1.1]pent-1-yl-4-{2,4-dichloro-6-[2-(4-chloro-1H-pyrazo-
l-1-yl)ethoxy]phenyl}-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxamid-
e
[0180] A solution of compound 1b (in 3 mL of DMF) and TEA (1 mL, 7
mmol) were added to a solution of compound 1a (51 mg, 0.11 mmol) in
DMF (2 mL). The mixture was heated at 70.degree. C. for 3 h. The
reaction was cooled to room temperature. Water (20 mL) was added to
the reaction mixture and EtOAc (2.times.50 mL) was added to extract
the aqueous solution. The combined organic layer was dried,
filtered, and concentrated to get a brown yellow oil. Isolation by
preparative HPLC gave compound 1 (23 mg, 0.043 mmol) as a white
solid in 39% yield. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm
1.94 (s, 6H), 2.35 (s, 1H), 3.72 (d, J=13.14 Hz, 1H), 3.96 (d,
J=13.14 Hz, 1H), 4.27-4.33 (m, 2H), 4.33-4.48 (m, 4H), 6.78 (s,
2H), 6.90 (s, 1H), 7.28 (d, J=1.77 Hz, 1H), 7.35 (d, J=1.52 Hz,
1H), 7.36 (s, 1H), 7.49 (s, 1H). LCMS (M+H).sup.+ 535.
[0181] Anal. Calcd for
C.sub.23H.sub.22Cl.sub.3N.sub.7O.sub.2.1H.sub.2O: C, 49.97; H,
4.38; N, 17.73. Found: C, 50.36; H, 4.23; N, 17.40.
##STR00014##
Compound 1a
4-{2,4-dichloro-6-[2-(4-chloro-pyrazol-1-yl)-ethoxy]-phenyl}-6,7-dihydro-5-
H-pyrrolo[3,4-d]pyrimidin-2-ylamine
[0182] Hydrogen chloride (0.5 mL, 2 mmol, 4M in dioxane) was added
to a solution of compound 1c (55.7 mg, 0.11 mmol) in
dichloromethane (10 mL). The mixture was stirred at room
temperature for 12 h and monitored by LC/MS. The solvent was
evaporated to afford compound 1a as a light yellow solid. This
crude residue was used for the synthesis of compound 1 without
further purification.
##STR00015##
Compound 1b
Imidazole-1-carboxylic acid bicyclo[1.1.1]pent-1-ylamide
[0183] Triethylamine (0.1 mL, 0.5 mmol) was added to a solution of
bicyclo[1.1.1]-pentan-1-amine hydrogen chloride (19 mg, 0.16 mmol)
and 1,1'-carbonyldiimidazole (32 mg, 0.192 mmol) in DMF (2 mL)
(clear solution turned into suspension). The suspension was used
for the synthesis of compound 1 without further isolation of
compound 1b.
Compound 1c
2-amino-4-{2,4-dichloro-6-[2-(4-chloro-pyrazol-1-yl)-ethoxy]-phenyl}-5,7-d-
ihydro-pyrrolo[3,4-d]pyrimidine-6-carboxylic acid tert-butyl
ester
[0184] Potassium carbonate (100 mg, 0.7 mmol) and compound 1e (120
mg, 0.5 mmol) were added sequentially to a solution of compound 1d
(98 mg, 0.25 mmol) in DMF (2 mL). The mixture was microwaved at
120.degree. C. for 40 min. Water (10 mL) and EtOAc (50 mL) were
added to the reaction mixture to stir. The organic layer was
collected, dried, filtered, and concentrated to get a yellowish
oil. This oil residue was purified by silica gel chromatography
(gradient elution 50.fwdarw.60% EtOAc in hexane) to give compound
1c (62.7 mg, 48.3% yield) as an oil. This oil was lyophilized to a
white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm
1.38-1.48 (m, 9H), 3.63 (t, J=13.89 Hz, 1H), 3.91 (dd, J=24.00,
13.39 Hz, 1H), 4.28-4.34 (m, 2H), 4.40 (d, J=3.54 Hz, 4H), 6.81
(br. s., 2H), 7.27 (s, 1H), 7.32-7.34 (m, 1H), 7.39 (s, 1H), 7.55
(s, 1H). LCMS (M+H).sup.+ 527.
##STR00016##
Compound 1d
2-amino-4-(2,4-dichloro-6-hydroxy-phenyl)-5,7-dihydro-pyrrolo[3,4-d]pyrimi-
dine-6-carboxylic acid tert-butyl ester
[0185] Sodium carbonate (1.2 g, 11 mmol) in 5.6 mL of H.sub.2O was
added to a mixture of compound 1g (770 mg, 3.7 mmol) and compound
1f (1000 mg, 2.7 mmol) in 1,4-dioxane (45 mL). The mixture was
purged with nitrogen several times and then tetrakis
(triphenylphsophine) palladium (0) (270 mg) was added. The reaction
mixture and the resulting solution was heated to 80.degree. C. for
12 h. Water (50 mL) was added to the reaction mixture to quench the
reaction. EtOAc (2.times.100 mL) was then added to extract the
aqueous solution. The combined organic layer was dried, filtered,
and concentrated to get a brown oil. This oil residue was purified
by silica gel chromatography (gradient elution 30.fwdarw.40% EtOAc
in hexane) to give compound 1d (590 mg, 40% yield) as light brown
foam. This oil was lyophilized to a white solid. .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. ppm 1.37-1.47 (m, J=12.88 Hz, 9H),
4.07-4.24 (m, 2H), 4.42 (d, J=7.58 Hz, 2H), 6.82 (s, 2H), 6.95 (d,
J=1.77 Hz, 1H), 7.08-7.21 (m, 1H), 10.67 (s, 1H). LCMS (M+H).sup.+
398. Anal. Calcd for C.sub.17H.sub.18Cl.sub.2N.sub.4O.sub.3.0.5
CH.sub.2Cl.sub.2: C, 49.80; H, 4.36; N, 12.74. Found: C, 47.41; H,
4.46; N, 12.71.
##STR00017##
Compound 1e
Methanesulfonic acid 2-(4-chloro-pyrazol-1-yl)-ethyl ester
[0186] Sodium hydride (136 mg, 3.4 mmol, 60% dispersion in mineral
oil) was added to a solution of 4-chloropyrazole (342 mg, 3.3 mmol)
in DMF (10 mL) under N.sub.2. The mixture was stirred at room
temperature for 1 h. The reaction was transferred to
methanesulfonic acid 2-bromo-ethyl ester (820 mg, 4 mmol,
synthesized by following literature procedure, J. Med. Chem. 1983,
26(8), p1168) via a syringe. The mixture was stirred at 70.degree.
C. for 3 h. Water (30 mL) was added to the mixture and EtOAc
(2.times.100 mL) was added to extract the aqueous solution. The
combined organic layer was dried, filtered, and concentrated to get
a brown oil. This oil residue was purified by silica gel
chromatography (gradient elution 20.fwdarw.30% EtOAc in hexane) to
give compound 1e (59 mg, 8% yield) as a brown oil. .sup.1H NMR (400
MHz, CDCl.sub.3-d) .delta. ppm 2.89 (s, 3H), 4.38-4.43 (m, 2H),
4.55-4.61 (m, 2H), 7.49 (s, 1H), 7.49 (s, 1H).
Compound 1f
2-amino-4-iodo-5,7-dihydro-pyrrolo[3,4-d]pyrimidine-6-carboxylic
acid tert-butyl ester
[0187] To a suspension of ethyl
2-amino-4-chloro-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxylate
(compound I(C) when PG.sup.1 is EtOC(O)-- and X is Cl), (30.6 g,
126 mmol, 1.0 eq) in 750 mL acetonitrile was added
iodotrimethylsilane (100 mL, 703 mmol, 5.6 eq), and the resulting
reaction mixture was heated at reflux for 4 h. After cooling down
to ambient temperature, the reaction mixture was quenched with MeOH
(27 mL), and concentrated to dryness in vacuo. Traces of MeOH were
removed via coevaporation with toluene (150 mL). The resulting
residue was treated with Et.sub.2O (200 mL), and the precipitates
were filtered and washed with Et.sub.2O. The crude brown powder was
then stirred in refluxing EtOAc (250 mL), cooled to ambient
temperature and the solid was collected by filtration and washed
with ethyl acetate to afford the desired product as a brown powder
(52.2 g, .about.79%). This material was used for the next step
reaction without further purification. .sup.1H NMR (300 MHz,
DMSO-D6) .delta. ppm 9.47 (br s, 2H), 4.37 (s, 2H), 4.25 (s, 2H).
LCMS (M+H).sup.+: 263.2.
[0188] The above brown powder (52.2 g, .about.100 mmol) was
dissolved in dioxane (150 mL) and water (150 mL). In addition,
diisopropylethylamine (69.7 mL, 400 mmol) and Boc2O (43.6 g, 200
mmol) were added and the mixture warmed to 45.degree. C. The
mixture was stirred at ambient temperature and followed by NMR for
an additional 1.5 h. The product was poured into EtOAc (3.0 L) and
saturated NaHCO.sub.3 (aq.) (1.0 L), the layers were separated and
the aqueous layer was washed once more with EtOAc (1.0 L). The
combined organic layers were dried (Na.sub.2SO.sub.4), filtered and
concentrated in vacuo. The product was filtered over silica using
EtOAc (3 L) as an eluent, product not completely pure. Filtration
over a plug of silica using EtOAc (2 L) gave compound 1f (8 g, 22%)
as a tan solid. .sup.1H NMR (400 MHz, DMSO-D6) .delta. ppm 1.44 (d,
J=4.80 Hz, 9H), 4.25 (d, J=13.14 Hz, 2H), 4.42 (d, J=8.34 Hz, 2H),
7.06 (s, 2H). LCMS (M+H).sup.+: 348.2.
##STR00018##
I(C), where PG.sup.1 is EtOC(O)--, X is Cl
Preparation of Compound I(C)
##STR00019##
[0189] Step 1. ethyl N-(ethoxycarbonyl)-.beta.-alaninate (c)
[0190] Ethyl acrylate (a) (50 mL, 460 mmol, 1.1 eq), glycine ethyl
ester hydrochloride (b) (58.4 g, 418 mmol, 1 eq), and triethylamine
(58.3 mL, 418 mmol, 1 eq) in absolute EtOH (960 mL) was stirred at
ambient temperature for approximately 72 h. After reaction was
complete, volatile components were removed under vacuum and the
crude intermediate (c) was carried on directly.
Step 2. ethyl
N-(ethoxycarbonyl)-N-(2-ethoxy-2-oxoethyl)-.beta.-alaninate (e)
[0191] Crude intermediate (c) (418 mmol) was dissolved in
CH.sub.2Cl.sub.2 (275 mL) and triethylamine (58.3 mL, 418 mmol) was
added followed by ethyl chloroformate (d) (39.8 mL, 418 mmol). The
reaction was stirred at ambient temperature for about 24 h. After
the reaction was complete, the volatile components were removed
under vacuum. The crude product was then distilled under vacuum
(about 5 mm Hg) and dissolved in EtOAc which was washed with
aqueous saturated KHSO.sub.4.times.3, with brine.times.1 and dried
over Na.sub.2SO.sub.4. Following filtration, the volatile
components were removed under vacuum to afford intermediate (e) as
a clear oil (74.8 g, 272 mmol) in 65% yield over two steps.
Step 3. diethyl 4-oxopyrrolidine-1,3-dicarboxylate (f)
[0192] Intermediate (e) (18.0 g, 65.2 mmol) was added to an ice
bath cooled solution of NaOEt (32.6 mL) (21% by weight in EtOH) in
absolute EtOH (41.7 mL) under a nitrogen atmosphere. The ice bath
was removed and the mixture was heated at 80.degree. C. for about
12 h until the condensation was complete as observed by TLC. The
mixture was poured onto ice/water and extracted into EtOAc. The
solvent was dried with Na.sub.2SO.sub.4, filtered, and evaporated
to afford crude intermediate (f) as an off white solid (14.05 g)
which was carried on without purification.
Step 4. ethyl
2-amino-4-hydroxy-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxylate
(h)
[0193] A suspension of intermediate (f) (14.05 g) and guanidine
carbonate (g) (16.6 g, 91.9 mmol) was refluxed in t-butanol (147
mL) for about 6 h. The mixture was allowed to cool to ambient
temperature for about 2 h. The volatile components were removed
under vacuum and water was added. The pH was adjusted to about 6-7
using KHSO.sub.4. The resulting slurry was filtered to collect the
solids which were washed with water followed by EtOAc. The solids
were dried under vacuum to afford intermediate (h) as cream solids
(11.9 g, 53.1 mmol) in 87% yield. .sup.1H NMR (400 MHz, DMSO-D6)
.delta. ppm 11.01 (s, 1H), 6.97 (s, 0.5H, possible tautomer), 6.70
(s, 2H), 4.25 (s, 4H), 4.13-4.03 (m, 2H), 1.22 (t, 3H). LCMS
(M+H).sup.+: 225.2.
Step 5. ethyl
2-amino-4-chloro-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxylate
I(C)
[0194] Intermediate (h) (11 g, 49 mmol) was azeotroped.times.2 with
toluene. Anhydrous acetonitrile (250 mL) and POCl.sub.3 (25 mL, 270
mmol) were added and the mixture was refluxed for about 2.5 h.
Additional POCl.sub.3 (50 mL) was added and the mixture was
refluxed for an additional 2 h. The volatile components were
concentrated under vacuum at 40.degree. C. to give a red solution.
A minimum amount of dry acetonitrile was added until the solution
was readily transferable whereupon it was poured onto ice in a
large beaker. The flask was further rinsed with a small amount of
acetonitrile which was added to the ice. Water (about 50 mL) was
added to the ice mixture to help it with stirring. Concentrated
NH.sub.4OH (25 mL) was added slowly with stirring until the ice
slurry mixture was strongly basic, then 50% aqueous NaOH (25 mL)
was also added to the still stirring slurry of ice. Additional ice
was added. After about 5 minutes stirring as ice slurry, EtOAc was
added. After stirring in the beaker for several more minutes, water
was added to help melt the ice. The mixture was poured into a
separatory funnel and the layers were allowed to partition. The
aqueous layer was extracted with EtOAc.times.3. The combined EtOAc
extracts were washed .times.2 with saturated aqueous KHSO.sub.4,
.times.2 with saturated aqueous NaHCO.sub.3, .times.1 with brine,
dried over Na.sub.2SO.sub.4, filtered and evaporated to afford a
pale pink powder which was triturated with ethyl acetate to give
compound I(C) as pale pink solids (6.8 g, 28 mmol) in 57% yield.
HPLC/LCMS purity was greater than 90%. .sup.1H NMR (400 MHz,
DMSO-D6) .delta. ppm 7.20 (s, 2H), 4.48 (s, 2H), 4.45 (s, 2H),
4.17-4.08 (m, 2H), 1.24 (t, 3H). LCMS (M+H).sup.+: 243.2,
245.2.
Preparation of Compound 1g
2,4-dichloro-6-hydroxyphenylboronic acid
##STR00020##
[0195] Preparation of Compound a:
[0196] To a solution of 3,5-dichloro-phenol (70 g, 0.43 mol) in dry
toluene (1 L) was added NaH (51.5 g, 1.29 mol) portionwise at
0.degree. C. under N.sub.2 atmosphere. After the addition, the
resulting mixture was allowed to warm up to room temperature and
stirred for 20 minutes. The suspension was then cooled back to
0.degree. C., and iodine (253.81 g, 91.5 mol) was added slowly.
Then the reaction mixture was stirred at room temperature
overnight. TLC (petroleum ether/CH.sub.2Cl.sub.2 1:1) indicated
complete consumption of starting material. The reaction mixture was
quenched with 1 N HCl (1 L) and diluted with ether (1 L). The
separated organic layer was washed with brine (500 mL), dried over
Na.sub.2SO.sub.4 and concentrated in vacuo to give crude compound
2, which was purified by column chromatography (silica gel,
petroleum ether/CH.sub.2Cl.sub.2 from 5:1 to 1:1) to yield pure
compound a (85 g, yield: 68%) as a white solid.
Preparation of Compound b:
[0197] A mixture of compound a (67 g, 0.23 mol),
chloromethoxy-ethane (31.8 g, 0.29 mol) and Cs.sub.2CO.sub.3 (63.7
g, 0.2 mol) in DMF (600 mL) was stirred at room temperature for 2
hours. TLC (petroleum ether/EtOAc 2:1) indicated complete
consumption of compound a. The reaction mixture was washed with
H.sub.2O (500 mL.times.3) and brine (500 mL), dried over
Na.sub.2SO.sub.4 and concentrated in vacuo to give crude compound
3, which was purified via column chromatography (silica gel,
EtOAc/hexane 1:50) to yield pure compound b (80 g, 100%) as a
yellow solid.
Preparation of Compound c:
[0198] A solution of compound b (77 g, 0.22 mol),
4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (57 g, 0.44 mol) and
Et.sub.3N (92 mL, 0.66 mol) in dioxane (500 mL) was purged with
N.sub.2 for 30 minutes. Then Pd(OAc).sub.2 (2.7 g, 0.011 mol) and
biphenyl-2-yl-dicyclohexyl-phosphane (8.5 g, 0.022 mol) were added
to the resulting mixture. After the addition, the reaction mixture
was stirred at 80.degree. C. for 1.5 hours. TLC (petroleum
ether/EtOAc 30:1) indicated complete consumption of compound b. The
resulting mixture was washed with saturated NH.sub.4Cl (500 mL),
H.sub.2O (500 mL) and brine (500 mL) in sequence, dried over
Na.sub.2SO.sub.4 and concentrated in vacuo to give crude compound
c, which was purified by column chromatography (silica gel,
petroleum ether/EtOAc from 5:1 to 1:1) to yield pure compound c (35
g, yield: 45%) as a brown solid. Preparation of compound 1g: To a
stirred solution of compound c (35 g, 0.1 mol) in dry
CH.sub.2Cl.sub.2 (200 mL) was added BBr.sub.3 (125 g, 0.5 mol)
dropwise at 0.degree. C. under N.sub.2 atmosphere. After stirring
for 20 minutes, the reaction mixture was poured into ice water,
basified to pH .about.10 by 3 N NaOH (100 mL) and the organic layer
was separated. The separated aqueous layer was adjusted to pH
.about.3 with 1 N HCl (500 mL) and extracted with EtOAc (500
mL.times.3), the combined organic layers were washed with brine
(1.0 L), dried over Na.sub.2SO.sub.4 and concentrated in vacuo to
yield compound 1g (39.7 g, yield: 80%) as a white solid. .sup.1H
NMR (400 MHz, MeOD): .delta. 6.875-6.878 (d, 1H), 6.727-6.737 (d,
1H).
Example 2
2-amino-N-cyclopropyl-4-{2,4-dichloro-6-[2-(4-chloro-1H-pyrazol-1-yl)ethox-
y]phenyl}-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxamide
[0199] Potassium carbonate (46 mg, 0.33 mmol) and compound 2b and
2c (82 mg, 0.5 mmol) were added sequentially to a solution of
compound 2a (42 mg, 0.11 mmol) in DMF (3 mL). The mixture was
heated at 120.degree. C. for 40 min in a microwave. Water (10 mL)
and EtOAc (50 mL) were added to the reaction mixture and stirred.
The organic layer was collected, dried, filtered, and concentrated
to get a yellowish oil. This oil residue was purified by
preparative HPLC to give compound 2 (31.5 mg, 56% yield) as a white
solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 0.35-0.45
(m, 2H), 1.84 (s, 1H), 3.69 (d, J=13.39 Hz, 1H), 3.95 (dd, J=13.01,
1.39 Hz, 1H), 4.13-4.47 (m, 6H), 6.36 (s, 1H), 6.77 (s, 2H), 7.27
(d, J=1.77 Hz, 1H), 7.33 (d, J=1.77 Hz, 1H), 7.35 (s, 1H), 7.48 (s,
1H). LCMS (M+H).sup.+ 510.
[0200] Anal. Calcd for
C.sub.21H.sub.20Cl.sub.3N.sub.7O.sub.2.1.25H.sub.2O: C, 47.47; H,
4.27; N, 18.45. Found: C, 47.13; H, 4.01; N, 18.72.
##STR00021##
Compound 2a
2-amino-4-(2,4-dichloro-6-hydroxy-phenyl)-5,7-dihydro-pyrrolo[3,4-d]pyrimi-
dine-6-carboxylic acid cyclopropylamide
[0201] Sodium carbonate solution (7.8 mL, 2M, 9.2 mmol) were added
to a solution of compound 1g (668 g, 3.2 mmol) and compound
III(B)-1 (1.1 g, 3.1 mmol) in 1,4-dioxane (30 mL). The mixture was
purged with N2 for 15 min, then tetrakis (triphenylphsophino)
palladium (0) (355 mg, 0.3 mmol) was added. The resulting mixture
was stirred at 80.degree. C. for 12 h. The reaction mixture was
filtered through Celite pad and washed well with MeOH. The filtrate
was concentrated by vacuum. The residue was partitioned between
EtOAc (2.times.500 mL) and brine (100 mL). The organic layer was
dried, filtered, and concentrated to get a yellow oil. This yellow
oil was treated with CH.sub.2Cl.sub.2 and hexane and the
precipatate was collected and washed well with hexane to give
compound 2a (1.1 g, 97%). .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 0.33-0.44 (m, 2H), 0.46-0.59 (m, 2H), 0.80-0.91 (m,
1H), 4.11 (br. s., 2H), 4.39 (s, 2H), 6.45 (d, J=2.78 Hz, 1H),
6.74-6.84 (m, 2H), 6.91-7.00 (m, 1H), 7.14 (d, J=1.77 Hz, 1H),
10.64 (br. s., 1H). LCMS (M+H).sup.+: 380.
##STR00022##
Compound III(B)-1
2-amino-4-iodo-5,7-dihydro-pyrrolo[3,4-d]pyrimidine-6-carboxylic
acid cyclopropylamide
[0202] Iodotirmethylsilane (25 mL, 176 mmol) was added to a
suspension of compound I(C) (8.36 g, 24.7 mmol) in ACN (200 mL) at
room temperature. The mixture was refluxed at 90.degree. C. for 3
h. The reaction mixture was cooled to rt, and then quenched with
MeOH (10 mL), concentrated by vacuum. The residue was treated with
Et2O (100 mL), and the precipate was collected by filtration and
washed well with ether to give the HI salt of
4-Iodo-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-2-ylamine as a brown
color solid (14.4 g, 81%). This crude product was ready to use for
next reaction without further purification.
[0203] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 4.25 (t,
J=4.80 Hz, 1H), 4.29-4.47 (m, 3H), 6.02 (br. s., 2H), 9.42 (br. s.,
1H). LCMS (M+H).sup.+: 263.
A solution of compound 10a (in 5 mL of DMF) and TEA (2.8 mL, 20
mmol) were added to a solution of
4-Iodo-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-2-ylamine HI salt
(2590 mg, 5 mmol) in DMF (10 mL). The mixture was heated at
65.degree. C. for 2 h. The reaction was cooled down to room
temperature. Water (50 mL) was added to the reaction mixture and
EtOAc (2.times.100 mL) was added to extract the aqueous solution.
The combined organic layer was dried, filtered, and concentrated to
get a brown yellow oil. This oil residue was purified by silica gel
chromatography (gradient elution 0.fwdarw.10% CH.sub.3OH in
dichloromethane) to give compound III(B)-1 as a pale yellow solid
(1.1 g, 62% yield). .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm
0.38-0.48 (m, 2H), 0.51-0.64 (m, 2H), 2.53-2.59 (m, 1H), 4.24 (s,
2H), 4.36-4.44 (m, 2H), 6.56 (d, J=2.78 Hz, 1H), 6.96-7.10 (m, 2H).
LCMS (M+H).sup.+: 346.0.
##STR00023##
Preparation of Compounds 2b and 2c
4-chloro-1-(2-bromo-ethyl)-1H-pyrazole and
4-Chloro-1-(2-chloro-ethyl)-1H-pyrazole
[0204] Sodium hydride (293 mg, 7.3 mmol, 60% dispersion in mineral
oil was added to a solution of chloropyrazole (500 mg, 4.9 mmol) in
DMF (8 mL) at room temperature. The mixture was stirred at room
temperature for 40 min. The mixture was then canulated to a
1-bromo-2-chloroethane (856 mg, 5.9 mmol, in 1 mL of DMF). The
mixture was heated to 60.degree. C. for 12 h. Water (20 mL) was
added to the reaction mixture and EtOAc (2.times.50 mL) was added
to extract the aqueous solution. The combined organic layer was
dried, filtered, and concentrated to get a waxy oil residue. LC/MS
and .sup.1H-NMR indicated it's the mixture of two products,
compound 2b and compound 2c (498 mg, 61.9% yield).
Example 3
2-amino-N-bicyclo[1.1.1]pent-1-yl-4-{2,4-dichloro-6-[2-(4-chloro-1H-pyrazo-
l-1-yl)ethoxy]phenyl}-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxamid-
e
[0205] A solution of compound 1b (in 3 mL of DMF) and TEA (1 mL, 7
mmol) were added to a solution of compound 3a (50 mg, 0.11 mmol) in
DMF (2 mL). The mixture was heated at 70.degree. C. for 3 h. The
reaction was cooled down to room temperature. Water (20 mL) was
added to the reaction mixture and EtOAc (2.times.50 mL) was added
to extract the aqueous solution. The combined organic layer was
dried, filtered, and concentrated to get a brown yellow oil.
Isolation by preparative HPLC gave compound 2 (34 mg, 0.072 mmol)
as a white solid in 63% yield. .sup.1H NMR (400 MHz, dmso-d.sub.6)
.delta. ppm 1.89-1.97 (m, 6H), 1.99 (s, 3H), 2.26-2.39 (m, 1H),
3.49 (d, J=12.38 Hz, 1H), 3.82 (dd, J=12.76, 1.89 Hz, 1H),
4.19-4.46 (m, 6H), 6.01 (t, J=2.02 Hz, 1H), 6.67 (s, 2H), 6.82 (s,
1H), 6.99 (s, 1H), 7.02 (d, J=1.52 Hz, 1H), 7.19 (d, J=2.02 Hz,
1H), 7.30 (d, J=1.26 Hz, 1H). LCMS (M+H).sup.+ 481.
##STR00024##
Compound 3a
4-[4-chloro-2-methyl-6-(2-pyrazol-1-yl-ethoxy)-phenyl]-6,7-dihydro-5H-pyrr-
olo[3,4-d]pyrimidin-2-ylamine (110646-545)
[0206] Hydrogen chloride (0.5 mL, 2 mmol, 4M in dioxane) was added
to a solution of compound 3b (58 mg, 0.12 mmol) in dichloromethane
(10 mL). The mixture was stirred at room temperature for 12 h and
monitored by LC/MS. The solvent was evaporated to afford compound
3a as a light yellow solid residue. This crude residue was used for
the synthesis of compound 1 without further purification.
##STR00025##
Compound 3b
2-amino-4-[2,4-dichloro-6-(2-pyrazol-1-yl-ethoxy)-phenyl]-5,7-dihydro-pyrr-
olo[3,4-d]pyrimidine-6-carboxylic acid tert-butyl ester
[0207] Potassium carbonate (100 mg, 0.7 mmol) and
1-(2-Bromo-ethyl)-1H-pyrazole (synthesized followed the literature
procedure: Org. Letters, 2006, 8(10), p. 2043) (67 mg, 0.38 mmol)
were added sequentially to a solution of compound 3c (96 mg, 0.26
mmol) in DMF (2 mL). The mixture was microwaved at 120.degree. C.
for 40 min. Water (10 mL) and EtOAc (50 mL) were added to the
reaction mixture to stir. The organic layer was collected, dried,
filtered, and concentrated to get a yellowish oil. This oil residue
was purified by silica gel chromatography (gradient elution 100%
EtOAc) to give compound 3b (69.3 mg, 58% yield) as an oil. This oil
was lyophilized to a white solid. .sup.1H NMR (400 MHz,
dmso-d.sub.6) .delta. ppm 1.46 (s, 9H), 1.99 (s, 3H), 3.39-3.59 (m,
1H), 3.77 (d, J=22.48 Hz, 1H), 4.20-4.28 (m, 1H), 4.29-4.34 (m,
3H), 4.34-4.45 (m, 2H), 6.03 (q, J=2.02 Hz, 1H), 6.70 (s, 2H), 6.98
(s, 1H), 7.03 (s, 1H), 7.24 (dd, J=4.80, 2.02 Hz, 1H), 7.31 (dd,
J=15.92, 1.52 Hz, 1H). LCMS (M+H).sup.+ 472.
##STR00026##
Compound 3c
2-amino-4-(2,4-dichloro-6-hydroxy-phenyl)-5,7-dihydro-pyrrolo[3,4-d]pyrimi-
dine-6-carboxylic acid tert-butyl ester
[0208] Sodium carbonate (512 mg, 4.8 mmol) in 2.4 mL of H.sub.2O
was added to a mixture of compound 3d (300 mg, 1.6 mmol) and
compound 1f (583 mg, 1.6 mmol) in 1,4-dioxane (10 mL). The mixture
was purged with nitrogen several times and then tetrakis
(triphenylphosphino) palladium (0) (116 mg) was added. The reaction
mixture and the resulting solution was heated at 120.degree. C. for
40 min in microwave. Water (50 mL) was added to the reaction
mixture to quench the reaction. EtOAc (2.times.100 mL) was then
added to extract the aqueous solution. The combined organic layer
was dried, filtered, and concentrated to get a brown oil. This oil
residue was purified by silica gel chromatography (gradient elution
60.fwdarw.70% EtOAc in hexane) to give compound 3c (361 mg, 59%
yield) as light brown foam.
[0209] 1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.38-1.46 (m,
9H), 2.03 (s, 3H), 4.05-4.48 (m, 4H), 6.71 (s, 2H), 6.80 (s, 1H),
6.82 (s, 1H), 10.07 (s, 1H). LCMS (M+H).sup.+ 378.
##STR00027##
Compound 3d
5-chloro-2,3-dimethyl-phenolboronic acid
##STR00028##
[0210] Step#1 procedure: To a clear solution of SM (5.46 mL, 45
mmol, 1.0 eq) in DCM (300 mL) at ice-NaCl bath was added
Ipy.sub.2BF.sub.4 (20.3 g, 53.4 mmol, 1.20 eq). The resulting
solution was stirred at the 0.degree. C. bath and was then warmed
slowly to rt. The reaction turned slowly from orange solution to
orange suspension. The reaction was then stirred at rt overnight.
The reaction was diluted with DCM (200 mL), and washed with aqueous
saturated Na.sub.2S.sub.2O.sub.3. The organic layer was collected,
dried over Na.sub.2SO.sub.4, filtered, and concentrated. The
residue was columned on silica gel to afford 10.50 g of the desired
product as a brown solid in 88% yield. .sup.1H NMR (400 MHz,
CDCl.sub.3-d) .delta. ppm 4.05 (br. s., 2H), 7.02 (d, J=2.27 Hz,
1H), 7.50 (d, J=2.27 Hz, 1H).
[0211] Step#2 procedure: A mixture of SM (1.00 g, 3.74 mmol, 1.0
eq), CuI (73 mg, 0.374 mmol, 0.10 eq), 1,10-phenanthroline (139 mg,
0.748 mmol, 0.20 eq), Cs.sub.2CO.sub.3 (2.49 g, 7.48 mmol, 2.0 eq),
MeOH (10 mL) was reacted under Microwave conditions (120.degree.
C., 2 h, stirring). The LC-MS indicated that the reaction was
complete and the majority of the compound is the desired product.
The reaction was repeated ten times, each time in 1 gram scale. All
these four reaction was combined, diluted with MeOH and EtOAc. The
mixture was passed through celite to remove insoluble stuff. The
black filtrate was concentrated on the reduced pressure. The
residue was column on silica gel using 9:1 to 4:1 heptane/EtOAc to
afford 4.0 g (totally four run) of the desired product as a brown
solid in 62.3% yield.
Step#3 procedure: At 0.degree. C., to a suspension of SM (2.68 g,
15.6 mmol, 1.0 eq) in 25 mL conc. HCl (37%) was added a solution of
NaNO.sub.2 (2.27 g, 31.2 mmol, 2.0 eq) in 25 mL water. After being
stirred at 0.degree. C. for 10 min, an orange suspension was
obtained forming diazonium salts compound. A solution of KI (10.4
g, 62.5 mmol, 4.0 eq) in water (50 mL) was added into the diazonium
salt. During the addition, lots of solid stuff was generated.
[0212] The reaction was then stirred overnight at rt. After being
stirred at rt overnight, the reaction was diluted with 300 mL
EtOAc. The organic layer was collected and washed with 1.0 M
Na.sub.2S.sub.2O.sub.3. The organic layer was then dried over
Na.sub.2SO.sub.4, filtered, and concentrated. The residue was
columned on ISCO (10% EtOAc in Heptane) to afford 3.30 g of the
desired product in 75% yield as a yellow solid. .sup.1H NMR (400
MHz, CDCl.sub.3-d) .delta. ppm 2.45 (s, 3H), 3.88 (s, 3H), 6.64 (d,
J=1.76 Hz, 1H), 6.91 (d, J=1.76 Hz, 1H).
Step#4 procedure: To a 20 mL Microwave vial were added
Pd(OAc).sub.2 (39.7 mg, 0.177 mmol, 0.10 eq), SM (500 mg, 1.77
mmol, 1.0 eq), dioxane (12 mL), Pinacol borane (0.514 mL, 3.54
mmol, 2.0 eq), Et.sub.3N (0.74 mL, 5.31 mmol, 3.0 eq), and
phosphine ligand (124 mg, 0.354 mmol, 0.20 eq). The mixture was
purged with N2 and reacted in Microwave 120.degree. C. for 30 min.
The reaction was diluted with EtOAc (100 mL), filtered through
celite. The filtrate was washed with brine (100 mL). The organic
layer was dried and concentrated. The residue was columned on ISCO
using 10% EtOAc/Heptane to afford 4.00 g of the desired product in
80% yield as a white yellow solid. .sup.1H NMR (400 MHz,
CDCl.sub.3-d) .delta. ppm 1.38 (s, 12H), 2.32 (s, 3H), 3.76 (s,
3H), 6.64 (d, J=1.51 Hz, 1H), 6.76 (d, J=1.01 Hz, 1H). Step#5
procedure: To a solution of SM (1.50 g, 5.02 mmol) in 20 anhydrous
dichloromethane at 0.degree. C. was added Boron tribromide (12.6
mL, 12.6 mml, 2.5 eq, 1.0 M in DCM) slowly over 5 minutes. After
stirring for 15 minutes at 0.degree. C., the reaction was complete
by HPLC, and was poured into 20 mL ice water. The biphasic mixture
was stirred vigorously, and the aqueous phase was brought to
pH=.about.10 with 1 M NaOH (10 mL). The organic layer was
separated, and discarded. The aqueous phase was acidified to
pH=.about.3 with 1 M HCl, white precipitates were generated,
filtered, and dried at 60.degree. C. under house vacuum to afford
500 mg (53% yield) of the desired product as a white solid.
Example 4
2-amino-N-bicyclo[1.1.1]pent-1-yl-4-{2,4-dichloro-6-[2-(4-fluoro-1H-pyrazo-
l-1-yl)ethoxy]phenyl}-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxamid-
e
[0213] A solution of compound 1b (in 3 mL of DMF) and TEA (1 mL, 7
mmol) were added to a solution of compound 4a (200 mg, 0.49 mmol)
in DMF (5 mL). The mixture was heated at 70.degree. C. for 3 h. The
reaction was cooled down to room temperature. Water (30 mL) was
added to the reaction mixture and EtOAc (2.times.100 mL) was added
to extract the aqueous solution. The combined organic layer was
dried, filtered, and concentrated to get a brown yellow oil. This
oil residue was purified by silica gel chromatography (gradient
elution 0.fwdarw.10% CH.sub.3OH in EtOAc) to give compound 4 (161
mg, 63.5% yield) as an oil. This oil was lyophilized to a white
solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.94 (s,
6H), 2.34 (s, 1H), 3.65 (d, J=13.14 Hz, 1H), 3.93 (d, J=13.14 Hz,
1H), 4.20-4.28 (m, 2H), 4.30-4.43 (m, 4H), 6.79 (s, 2H), 6.89 (br.
s., 1H), 7.23-7.29 (m, 2H), 7.34 (d, J=1.77 Hz, 1H), 7.36 (d,
J=4.55 Hz, 1H). LCMS (M+H).sup.+ 519.
[0214] Anal. Calcd for
C.sub.23H.sub.22Cl.sub.2FN.sub.7O.sub.2.1H.sub.2O: C, 51.50; H,
4.51; N, 18.28. Found: C, 51.81; H, 4.34; N, 18.02.
##STR00029##
Compound 4a
4-{2,4-dichloro-6-[2-(4-fluoro-pyrazol-1-yl)-ethoxy]-phenyl}-6,7-dihydro-5-
H-pyrrolo[3,4-d]pyrimidin-2-ylamine
[0215] Hydrogen chloride (2.4 mL, 9.8 mmol, 4M in dioxane) was
added to a solution of compound 4b (249 mg, 0.49 mmol) in
CH.sub.3OH (10 mL). The mixture was stirred at room temperature for
12 h and monitored by LC/MS. The solvent was evaporated to afford
compound 4a as a light yellow solid residue. This crude residue was
used for the synthesis of compound 1 without further
purification.
##STR00030##
Compound 4b
2-amino-4-{2,4-dichloro-6-[2-(4-fluoro-pyrazol-1-yl)-ethoxy]-phenyl}-5,7-d-
ihydro-pyrrolo[3,4-d]pyrimidine-6-carboxylic acid tert-butyl
ester
[0216] Potassium carbonate (282 mg, 2 mmol) and compound 4c (674
mg, 1.4 mmol) were added sequentially to a solution of compound 1d
(270 mg, 0.68 mmol) in DMF (8 mL). The mixture was heat at
120.degree. C. for 40 min in microwave. Water (20 mL) and EtOAc
(2.times.50 mL) were added to the reaction mixture to stir. The
organic layer was collected, dried, filtered, and concentrated to
get a yellowish oil. This oil residue was purified by silica gel
chromatography (gradient elution 90.fwdarw.100% EtOAc in hexane) to
give compound 4b (194 mg, 56% yield) as a pale yellow foam. .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.34-1.50 (m, 9H),
3.48-3.64 (m, J=6.06 Hz, 1H), 3.79-3.97 (m, 1H), 4.21-4.31 (m, 2H),
4.30-4.50 (m, 4H) 6.83 (s, 2H), 7.27 (s, 1H), 7.30-7.38 (m, 2H),
7.42 (dd, J=10.86, 4.55 Hz, 1H). LCMS (M+H).sup.+ 509.
##STR00031##
Synthesis of compounds 4c and 4d
4-fluoro-1-(2-bromo-ethyl)-1H-pyrazole and
4-fluoro-1-(2-chloro-ethyl)-1H-pyrazole
[0217] Sodium hydride (700 mg, 17.4 mmol, 60% dispersion in mineral
oil was added to a solution of fluoropyrazole (1000 mg, 11.6 mmol)
in DMF (7 mL) at room temperature. The mixture was stirred at room
temperature for 40 min. The mixture was then canulated to a
1-bromo-2-chloroethane (2000 mg, 13.9 mmol, in 1 mL of DMF). The
mixture was heated to 60.degree. C. for 12 h. Water (50 mL) was
added to the reaction mixture and THF (2.times.200 mL) was added to
extract the aqueous solution. The combined organic layer was dried,
filtered, and concentrated to get a waxy oil residue. LC/MS and
.sup.1H-NMR indicated it's the mixture of two products, compound 4c
and compound 4d (1000 mg, 57.4% yield). Synthesis of
fluoropyrazole: Fluoropyrazole was prepared and isolated by
modifying the method described in Organic Letters 1995, 3, p
239.
##STR00032##
Step#1 Procedure:
[0218] To a solution of compound 1 (750 g, 5.7 mol) and Et.sub.3N
(948 mL, 6.816 mol) in CH.sub.2Cl.sub.2 (2 L) was added TsCl
(1295.4 g, 6.8 mol) at 0.degree. C. in portions. The resulting
mixture was stirred at room temperature overnight. TLC (petroleum
ether/EtOAc=5:1) indicated the reaction was complete. Brine (5 L)
was added, and the mixture was extracted with CH.sub.2Cl.sub.2
(3.times.10 L). The combined organic layers were washed with brine
(2.times.5 L), dried over Na.sub.2SO.sub.4 and concentrated in
vacuo to give crude product, which was purified by column
chromatography (silica gel, petroleum ether/EtOAc from 20:1 to 5:1)
to yield compound 2 (1500 g, 92%) as a white solid.
Step#2 Procedure:
[0219] To a solution of compound 2 (100 g, 0.35 mmol) in THF (1.5
L) was added n-BuLi (336 ml, 0.8 mol) dropwise at -85.degree. C.
Then the mixture was stirred at -85.degree. C. for another 10
minutes. The reaction mixture was quenched with 3 N aqueous HCl
(300 mL) and allowed to warm to room temperature. The mixture was
basified to pH=7 with saturated aqueous NaHCO.sub.3 and extracted
with Et.sub.2O (3.times.5 L). The organic layer was dried over
Na.sub.2SO.sub.4 and concentrated in vacuum to give compound 3
(53.84 g, 58%) as a brown solid, which was used for the next step
directly.
[0220] Step#3 Procedure:
[0221] A solution of compound 3 (150 g, 0.5639 mol) in CH.sub.3CN
(500 mL) was added to a solution of NHEt.sub.2 (50 g, 0.7 mol),
TBAF (56.3 mL, 0.05639 mol) and Et.sub.3N (78 mL, 0.6 mol) in
CH.sub.3CN (300 mL) at 0.degree. C. The resulting solution was
stirred at room temperature overnight. TLC (petroleum
ether/EtOAc=1:1) indicated the reaction was complete. The mixture
was diluted with brine (300 mL) and extracted with EtOAc (500
mL.times.3). The combined organic layers were dried over
Na.sub.2SO.sub.4 and concentrated in vacuum to give crude product,
which was purified by column chromatography (silica gel, petroleum
ether/EtOAc=1:1) to yield compound 4 (60 g, 73%) as brown oil.
Step#4 Procedure:
[0222] A mixture of compound 4 (100 g, 0.7 mol) and
NH.sub.2NH.sub.2.2HCl (79.6 g, 0.8 mol) in EtOH (200 mL) and H2O
(150 mL) was stirred at 78.degree. C. for 4 hours. TLC (petroleum
ether/EtOAc=1:1) indicated the reaction was complete. The reaction
mixture was allowed to cool to room temperature. The mixture was
basified to pH=7 with saturated aqueous NaHCO.sub.3 and extracted
with EtOAc (3.times.2 L). The combined organic layers were dried
over Na.sub.2SO.sub.4 and concentrated in vacuum to give crude
product, which was purified by column chromatography (silica gel,
petroleum ether/EtOAc=1:1) to yield fluoropyrazole (36.25 g, 61%)
as a brown solid.
[0223] .sup.1H NMR (400 MHz, dmso-d.sub.6) .delta. ppm 7.64 (br.
s., 2H), 12.61 (br. s., 1H).
Example 5
2-amino-N-bicyclo[1.1.1]pent-1-yl-4-{2,4-dichloro-6-[2-(4-fluoro-1H-pyrazo-
l-1-yl)ethoxy]phenyl}-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxamid-
e
[0224] A solution of compound 5a (in 3 mL of DMF) and TEA (1 mL, 7
mmol) were added to a solution of compound 4a (195 mg, 0.48 mmol)
in DMF (4 mL). The mixture was heated at 70.degree. C. for 3 h. The
reaction was cooled down to room temperature. Water (30 mL) was
added to the reaction mixture and EtOAc (2.times.50 mL) was added
to extract the aqueous solution. The combined organic layer was
dried, filtered, and concentrated to get a brown yellow oil. This
oil residue was purified by silica gel chromatography (gradient
elution 0.fwdarw.10% CH.sub.3OH in EtOAc) to give compound 5 (192
mg, 75.5% yield) as an oil. This oil was lyophilized to a white
solid. .sup.1H NMR (400 MHz, dmso-d.sub.6) .delta. ppm 3.66 (d,
J=13.14 Hz, 1H), 3.82 (br. s., 2H), 3.98 (d, J=12.63 Hz, 1H), 4.26
(d, J=4.04 Hz, 2H), 4.30-4.38 (m, 2H), 4.42 (br. s., 2H), 6.83 (s,
2H), 6.99 (s, 1H), 7.23 (d, J=4.29 Hz, 1H), 7.29 (d, J=1.52 Hz,
1H), 7.35 (d, J=1.77 Hz, 1H), 7.37 (d, J=4.55 Hz, 1H). LCMS
(M+H).sup.+ 534.
[0225] Anal. Calcd for
C.sub.20H.sub.17Cl.sub.2F.sub.4N.sub.7O.sub.2.0.25H.sub.2O: C,
44.58; H, 3.27; N, 18.20. Found: C, 44.87; H, 3.34; N, 17.86.
##STR00033##
Compound 5a
1-[(Z)-methyliminomethyl]-3-(2,2,2-trifluoro-ethyl)-1-vinyl-urea
[0226] TEA (1.3 mL, 0.8 mmol) was added to a solution of
2,2,2-trifluoroethylamine (72 mg, 1 mmol) and
1,1'-carbonyldiimidazole (135 mg, 0.8 mmol) in DMF (4 mL) (clear
colorless solution). The solution was used for the synthesis of
compound 5 without further isolation of compound 5a.
Example 6
2-amino-N-bicyclo[1.1.1]pent-1-yl-4-{2,4-dichloro-6-[2-(4-fluoro-1H-pyrazo-
l-1-yl)ethoxy]phenyl}-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxamid-
e
[0227] Potassium carbonate (120 mg, 0.85 mmol) and compound 6b (89
mg, 0.4 mmol) were added sequentially to a solution of compound 6a
(71 mg, 0.17 mmol) in DMF (3 mL). The mixture was heated at
100.degree. C. for 60 min in microwave. Water (20 mL) and EtOAc
(100 mL) were added to the reaction mixture to stir. The organic
layer was collected, dried, filtered, and concentrated to get a
yellowish oil. This oil residue was purified by preparative HPLC to
give compound 6 (30 mg, 33% yield) as a white solid. .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. ppm 1.56 (t, J=18.95 Hz, 3H),
3.43-3.55 (m, 2H), 3.69 (d, J=13.14 Hz, 1H), 4.00 (d, J=12.88 Hz,
1H), 4.20-4.28 (m, 2H), 4.31-4.37 (m, 2H), 4.40-4.47 (m, 2H), 6.69
(s, 1H), 6.81 (s, 2H), 7.26 (d, J=4.29 Hz, 1H), 7.28 (d, J=1.77 Hz,
1H), 7.34 (d, J=1.77 Hz, 1H), 7.36 (d, J=4.80 Hz, 1H). LCMS
(M+H).sup.+ 530. Anal. Calcd for
C.sub.21H.sub.20Cl.sub.2F.sub.3N.sub.7O.sub.2.1.75H.sub.2O: C,
44.89; H, 4.22; N, 17.45. Found: C, 45.22; H, 3.90; N, 17.12.
##STR00034##
Compound 6a
2-amino-4-(2,4-dichloro-6-hydroxy-phenyl)-5,7-dihydro-pyrrolo[3,4-d]pyrimi-
dine-6-carboxylic acid (2,2-difluoro propyl)-amide
[0228] Sodium carbonate solution (7.8 mL, 2M, 15.5 mmol) were added
to a solution of compound 1g (1.07 g, 5.17 mmol) and compound
III(B)-2 (1.98 g, 5.17 mmol) in 1,4-dioxane (25 mL). The mixture
was purged with N2 for 15 min, then tetrakis (triphenylphsophino)
palladium (0) (597 mg, 0.517 mmol) was added. The resulting mixture
was stirred at 85.degree. C. for 3 hours. The reaction mixture was
filtered through Celite pad and washed well with MeOH. The filtrate
was concentrated by vacuum. The residue was partitioned between
EtOAc (2.times.500 mL) and brine (100 mL). The organic layer was
dried, filtered, and concentrated to get a brown oil. Isolation by
preparative HPLC to afford compound 6a as a white solid (305 mg,
14% yield).
[0229] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.54 (t,
J=19.07 Hz, 3H), 3.46 (br. s., 2H), 4.21 (br. s., 2H), 4.47 (s,
2H), 6.73-6.84 (m, 2H), 6.84-6.89 (m, 1H), 6.97 (d, J=1.77 Hz, 1H),
7.16 (d, J=2.02 Hz, 1H), 10.69 (br. s., 1H). LCMS (M+H).sup.+:
418.0, 420.0.
##STR00035##
Compound III(B)-2:
2-amino-4-iodo-5,7-dihydro-pyrrolo[3,4-d]pyrimidine-6-carboxylic
acid (2,2-difluoro-propyl)-amide
[0230] Iodotirmethylsilane (25 mL, 176 mmol) was added to a
suspension of compound I(C) (8.36 g, 24.7 mmol) in ACN (200 mL) at
room temperature. The mixture was refluxed at 90.degree. C. for 3
h. The reaction mixture was cooled to rt, and then quenched with
MeOH (10 mL), concentrated by vacuum. The residue was treated with
Et.sub.2O (100 mL), and the precipate was collected by filtration
and washed well with ether to give the Hi salt of
4-Iodo-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-2-ylamine as a brown
color solid (14.4 g, 81%). This crude product was ready to use for
next reaction without further purification.
[0231] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 4.25 (t,
J=4.80 Hz, 1H), 4.29-4.47 (m, 3H), 6.02 (br. s., 2H), 9.42 (br. s.,
1H). LCMS (M+H).sup.+: 263.0.
A solution of compound 9a (in 2 mL of DMF) and TEA (0.6 mL, 4.6
mmol) were added to a solution of
4-Iodo-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-2-ylamine HI salt
(590 mg, 1.1 mmol) in DMF (5 mL). The mixture was heated at
65.degree. C. for 2 h. The reaction was cooled down to room
temperature. Water (50 mL) was added to the reaction mixture and
EtOAc (2.times.100 mL) was added to extract the aqueous solution.
The combined organic layer was dried, filtered, and concentrated to
get a brown yellow oil. This oil residue was purified by silica gel
chromatography (gradient elution 0.fwdarw.100% EtOAc in
dichloromethane) to give compound III(B)-2 as a white solid (195
mg, 82% yield). .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm
1.50-1.60 (m, 3H), 3.51 (br. s., 2H), 4.32 (s, 2H), 4.46 (d, J=6.06
Hz, 2H). LCMS (M+H).sup.+: 384.0.
##STR00036##
Compound 6b
Methanesulfonic acid 2-(4-fluoro-pyrazol-1-yl)-ethyl ester
[0232] Methanesulfonyl chloride (16.8 g, 11.4 mL, 147 mmol) was
added to a solution of compound 6a (13.4 g, 98 mmol),
diisopropylamine (34.3 mL, 196 mmol), and
4-(dimethylamino)-pyridine (1.2 g, 9.78 mmol) in dichloromethane
(245 mL) in an ice bath. The reaction was warm to room temperature
and stirred at room temperature for 2 h. Water (50 mL) was added to
quench the reaction. The organic layer was dried, filtered, and
concentrated to get a brown oil. This oil residue was purified by
silica gel chromatography (0.fwdarw.60% EtOAc in hexane) to give
compound 6b (19.5 g, 96% yield) as a yellowish oil. .sup.1H NMR
(400 MHz, CDCl.sub.3-d) .delta. ppm 2.89 (s, 3H), 4.33-4.38 (m,
2H), 4.53-4.59 (m, 2H), 7.38 (d, J=1.52 Hz, 1H), 7.40 (d, J=1.01
Hz, 1H).
##STR00037##
Preparation of Compound 6c
2-(4-Fluoro-pyrazol-1-yl)-ethanol
[0233] Sodium hydride (13.8 g, 0.35 mol, 60% dispersion in mineral
oil) was added to a solution of fluoropyrazole (20 g, 0.23 mol) in
DMF (200 mL) 0.degree. C. The mixture was stirred at room
temperature for 1 h and then 2-bromo-ethanol (43 g, 0.35 mol) was
added dropwisely at 0.degree. C. The resulting mixture was stirred
at 40.degree. C. for 12 h. TLC (petroleum ether/EtOAc=1:1)
indicated the reaction was complete. The mixture was quenched with
saturated aqueous NH.sub.4Cl (200 mL). Then DMF was removed in
vacuo and the residue was partitioned between Et.sub.2O (1000 mL)
and H.sub.2O (1000 mL). The aqueous layer was extracted with more
Et.sub.2O (4.times.1 L). The combined organic layers were dried
over Na.sub.2SO.sub.4 and concentrated in vacuum to give compound
6c (20 g, 66%) as a yellow oil. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 3.69 (t, J=5.56 Hz, 2H), 4.04 (t, J=5.56 Hz, 2H), 4.90
(br. s., 1H), 7.43 (d, J=4.29 Hz, 1H), 7.81 (d, J=4.55 Hz, 1H).
##STR00038##
Example 7
2-amino-N-bicyclo[1.1.1]pent-1-yl-4-{4-chloro-2-[2-(4-fluoro-1H-pyrazol-1--
yl)ethoxy]-6-methylphenyl}-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carbo-
xamide
[0234] A solution of compound 1b (in 3 mL of DMF) and TEA (1 mL, 7
mmol) were added to a solution of compound 7a (186 mg, 0.48 mmol)
in DMF (5 mL). The mixture was heated at 70.degree. C. for 3 h. The
reaction was cooled down to room temperature. Water (50 mL) was
added to the reaction mixture and EtOAc (2.times.100 mL) was added
to extract the aqueous solution. The combined organic layer was
dried, filtered, and concentrated to get a brown yellow oil. This
oil residue was purified by silica gel chromatography (gradient
elution 0.fwdarw.10% CH.sub.3OH in EtOAc) to give compound 7 (195
mg, 82% yield) as an oil. This oil was lyophilized to a white
solid. .sup.1H NMR (400 MHz, MeOD) .delta. ppm 2.05 (s, 6H), 2.06
(s, 3H), 2.37 (s, 1H), 3.71 (d, J=12.63 Hz, 1H), 3.96 (dd, J=13.01,
1.39 Hz, 1H), 4.23-4.36 (m, 4H), 4.43-4.55 (m, 2H), 6.97 (s, 1H),
6.98 (s, 1H), 7.24 (d, J=4.55 Hz, 1H), 7.26 (d, J=4.29 Hz, 1H).
LCMS (M+H).sup.+ 499.
[0235] Anal. Calcd for
C.sub.24H.sub.25ClFN.sub.7O.sub.2.0.25H.sub.2O: C, 57.89; H, 5.06;
N, 19.69. Found: C, 57.31; H, 5.11; N, 19.44.
##STR00039##
Compound 7a
4-{4-chloro-2-[2-(4-fluoro-pyrazol-1-yl)-ethoxy]-6-methyl-phenyl}-6,7-dihy-
dro-5H-pyrrolo[3,4-d]pyrimidin-2-ylamine
[0236] Hydrogen chloride (2.4 mL, 9.5 mmol, 4M in dioxane) was
added to a solution of compound 7b (234 mg, 0.48 mmol) in
CH.sub.3OH (10 mL). The mixture was stirred at room temperature for
12 h and monitored by LC/MS. The solvent was evaporated to afford
compound 7a as a light yellow solid residue. This crude residue was
used for the synthesis of compound 7 without further
purification.
##STR00040##
Compound 7b
2-amino-4-{4-chloro-2-[2-(4-fluoro-pyrazol-1-yl)-ethoxy]-6-methyl-phenyl}--
5,7-dihydro-pyrrolo[3,4-d]pyrimidine-6-carboxylic acid tert-butyl
ester
[0237] Potassium carbonate (154 mg, 1.1 mmol) and compound 6b (120
mg, 0.6 mmol) were added sequentially to a solution of compound 3c
(84 mg, 0.22 mmol) in DMF (2 mL). The mixture was heated at
100.degree. C. for 40 min. in microwave. Water (10 mL) and EtOAc
(50 mL) were added to the reaction mixture to stir. The organic
layer was collected, dried, filtered, and concentrated to get a
yellowish oil. This oil residue was purified by silica gel
chromatography (90.fwdarw.100% EtOAc in hexane) to give compound 7b
(66 mg, 60% yield) as a light yellow foam. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 1.44 (s, 9H), 2.00 (s, 3H), 3.50 (dd,
J=23.49, 13.39 Hz, 1H), 3.83 (dd, J=25.52, 12.88 Hz, 1H), 4.17-4.31
(m, 4H), 4.33-4.41 (m, 2H), 6.70 (s, 2H), 6.99 (s, 1H), 7.04 (s,
1H), 7.28 (dd, J=18.44, 4.29 Hz, 1H), 7.42 (dd, J=10.74, 4.42 Hz,
1H). LCMS (M+H).sup.+ 489. Anal. Calcd for
C.sub.23H.sub.26ClFN.sub.6O.sub.3.0.5H.sub.2O.0.25
CH.sub.3CO.sub.2CH.sub.2CH.sub.3: C, 55.44; H, 5.62; N, 16.16.
Found: C, 55.78; H, 5.49; N, 15.93.
##STR00041##
Example 8
2-amino-4-{4-chloro-2-[2-(4-fluoro-1H-pyrazol-1-yl)ethoxy]-6-methylphenyl}-
-N-(2,2,2-trifluoroethyl)-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carbox-
amide
[0238] A solution of compound 5a (in 5 mL of DMF) and TEA (2 mL, 14
mmol) were added to a solution of compound 7a (504 mg, 1.3 mmol) in
DMF (5 mL). The mixture was heated at 70.degree. C. for 3 h. The
reaction was cooled down to room temperature. Water (50 mL) was
added to the reaction mixture and EtOAc (2.times.100 mL) was added
to extract the aqueous solution. The combined organic layer was
dried, filtered, and concentrated to get a brown yellow oil. This
oil residue was purified by silica gel chromatography (gradient
elution 0.fwdarw.10% CH.sub.3OH in EtOAc) to give compound 8 (470
mg, 70% yield) as an oil. This oil was lyophilized to a white
solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 2.01 (s,
3H), 3.61 (d, J=12.88 Hz, 1H), 3.75-3.85 (m, 2H), 3.91 (d, J=12.63
Hz, 1H), 4.26 (dd, J=12.88, 3.79 Hz, 4H), 4.36-4.44 (m, 2H), 6.73
(s, 2H), 6.92 (s, 1H), 7.01 (s, 1H), 7.06 (s, 1H), 7.23 (d, J=4.04
Hz, 1H), 7.37 (d, J=4.55 Hz, 1H). LCMS (M+H).sup.+ 514.
[0239] Anal. Calcd for
C.sub.21H.sub.20ClF.sub.4N.sub.7O.sub.2.1H.sub.2O: C, 47.42; H,
4.17; N, 18.43. Found: C, 47.54; H, 3.85; N, 18.13.
##STR00042##
Example 9
2-amino-4-{4-chloro-2-[2-(4-fluoro-1H-pyrazol-1-yl)ethoxy]-6-methylphenyl}-
-N-(2,2-difluoropropyl)-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxam-
ide
[0240] A solution of compound 9a (in 3 mL of DMF) and
diisopropylethylamine (1 mL) were added to a solution of compound
7a (136 mg, 0.35 mmol) in DMF (5 mL). The mixture was heated at
75.degree. C. for 2 h. The reaction was cooled down to room
temperature. Water (50 mL) was added to the reaction mixture and
EtOAc (2.times.100 mL) was added to extract the aqueous solution.
The combined organic layer was dried, filtered, and concentrated to
get a brown yellow oil. This oil residue was purified by silica gel
chromatography (gradient elution 0.fwdarw.10% CH.sub.3OH in EtOAc)
to give compound 9 (171 mg, 96% yield) as an oil. This oil was
lyophilized to a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 1.56 (t, J=18.95 Hz, 3H), 2.01 (s, 3H), 3.40-3.52 (m,
2H), 3.65 (d, J=13.14 Hz, 1H), 3.92 (d, J=12.63 Hz, 1H), 4.26 (dd,
J=11.62, 3.28 Hz, 4H), 4.37-4.45 (m, 2H) 6.66 (s, 1H), 6.70 (s, 2H)
7.01 (s, 1H), 7.06 (s, 1H), 7.26 (d, J=4.04 Hz, 1H), 7.36 (d,
J=4.55 Hz, 1H). LCMS (M+H).sup.+ 511.
[0241] Anal. Calcd for
C.sub.22H.sub.23ClF.sub.3N.sub.7O.sub.2.0.25H.sub.2O: C, 51.37; H,
4.60; N, 19.06. Found: C, 51.34; H, 4.65; N, 18.70.
##STR00043##
Compound 9a
Imidazole-1-carboxylic acid (2,2-difluoro-propyl)-amide
[0242] TEA (1.3 mL, 0.8 mmol) was added to a solution of
2,2-difluoropropylamine hydrochloride (82 mg, 0.62 mmol) and
1,1'-CARBONYLDIIMIDAZOLE (114 mg, 0.7 mmol) in DMF (3 mL) (clear
colorless solution). The solution was used for the synthesis of
compound 5 without further isolation of compound 9.
Example 10
2-amino-N-cyclopropyl-4-{2,4-dichloro-6-[2-(4-fluoro-1H-pyrazol-1-yl)ethox-
y]phenyl}-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxamide
[0243] Compound 10 was prepared in a manner similar to Example 9
except that imidazole-1-carboxylic acid cyclopropylamide (10a) was
substituted for imidazole-1-carboxylic acid
(2,2-difluoro-propyl)-amide (compound 9a). Compound 10 was obtained
as a white solid (26 mg, 40%). .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 0.36-0.44 (m, 2H), 0.49-0.63 (m, 2H), 1.19-1.28 (m,
1H), 3.65 (d, J=13.14 Hz, 1H), 3.93 (d, J=13.14 Hz, 1H), 4.21-4.28
(m, 2H), 4.29-4.44 (m, 4H), 6.35 (s, 1H), 6.78 (s, 2H), 7.20-7.29
(m, 2H), 7.33 (d, J=1.77 Hz, 1H), 7.35 (d, J=4.55 Hz, 1H). LCMS
(M+H).sup.+ 493. Anal. Calcd for
C.sub.21H.sub.20Cl.sub.2FN.sub.7O.sub.2.1.25H.sub.2O.0.25
CH.sub.3COOH: C, 48.74; H, 4.47; N, 18.50. Found: C, 48.75; H,
4.08; N, 18.24.
##STR00044##
Example 11
2-amino-4-{2,4-dichloro-6-[2-(4-cyano-1H-pyrazol-1-yl)ethoxy]phenyl}-N-(2,-
2-difluoropropyl)-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxamide
[0244] Compound 11b (136 mg, 0.88 mmol) was added to a mixture of
6a (147 mg, 0.35 mmol) and cesium carbonate (400 mg, 1.23 mmol) in
DMSO (3 mL). The resulting mixture was heated at 90.degree. C. for
12 h. Sat. sodium carbonate solution (50 mL) was added to the
mixture and EtOAc (2.times.50 mL) was added to extract the aqueous
solution. The combined organic layer was dried, filtered, and
concentrated to get a brown oil. Isolation by preparative HPLC gave
compound 11 (18 mg, 20%). .sup.1H NMR (400 MHz, DMSO-d6) .delta.
ppm 1.56 (t, J=19.07 Hz, 3H), 3.48 (d, J=6.06 Hz, 1H), 3.67 (br.
s., 1H), 3.92-4.08 (m, 2H), 4.36-4.50 (m, 6H), 6.70 (d, J=2.02 Hz,
1H), 6.78 (s, 2H), 7.31 (d, J=1.77 Hz, 1H), 7.34 (d, J=1.77 Hz,
1H), 7.81 (s, 1H), 8.19 (s, 1H). LCMS (M+H).sup.+ 537.
##STR00045##
Compound 11b
1-(2-chloro-ethyl)-1H-pyrazole-4-carbonitrile
[0245] Thionyl chloride (0.14 mL, 1.84 mmol) was added to a
suspension of compound 1c (160 mg, 0.92 mmol) in DCM (4 mL) at
.about.0.degree. C. (ice/water bath). The resulting mixture was
stirred at 0.degree. C. and warmed to room temperature for 90 min
while suspension become yellowish solution. The reaction mixture
was concentrated by vacuum. The residue was partitioned between
EtOAc (300 mL) and sat. NaHCO.sub.3 solution (50 mL) and brine (50
mL). The organic layer was dried (Na.sub.2SO.sub.4), filtered, and
concentrated by vacuum to give compound 11b as yellow grease (140
mg, 98%). This solid was used for the next step reaction without
further purification. .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm
3.96-4.08 (m, 2H), 4.47-4.56 (m, 2H), 8.12 (s, 1H), 8.64 (s,
1H).
##STR00046##
Preparation of Compound 11c
1-(2-Chloro-ethyl)-4-nitrosomethyl-1H-pyrazole
[0246] Hydroxyamine hydrochloride (114 mg, 1.6 mmol) was added to a
solution of compound 11d (166 mg, 1.1 mmol) and sodium acetate (174
mg, 2.1 mmol) in EtOH (4 mL). The resulting mixture was stirred at
90.degree. C. for 90 min. The reaction mixture was partitioned
between EtOAc (300 mL) and H.sub.2O (50 mL) and brine (50 mL). The
organic layer was dried, filtered, and concentrated to give
compound 11c as white solid (164 mg, 90%). .sup.1H NMR (400 MHz,
dmso-d6) .delta. ppm 4.00 (t, J=5.68 Hz, 2H), 4.49 (t, J=5.68 Hz,
2H), 7.36 (s, 1H), 7.85 (s, 1H), 8.33 (s, 1H), 11.26 (s, 1H). LCMS
(M+H).sup.+: 174
Preparation of Compound 11d
1-(2-Chloro-ethyl)-4-nitrosomethyl-1H-pyrazole
[0247] Compound 11d was prepared in a manner similar to compounds
4c and 4d except that 4-carbaldehydepyrazole was substituted for
4-fluoropyrazole in Example 4.
Example 12
2-amino-4-{2-[2-(4-bromo-1H-pyrazol-1-yl)ethoxy]-4,6-dichlorophenyl}-N-cyc-
lopropyl-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxamide
[0248] 4-Bromopyrazole (141 mg, 1 mmol) was added to a solution of
compound 12a (142 mg, 0.32 mmol), K.sub.2CO.sub.3 (265 mg, 1.92
mmol) and KI (106 mg, 0.64 mmol) in DMF (2 mL). The resulting
mixture was heated at 130.degree. C. 1 h in microwave. The reaction
mixture was partitioned between EtOAc (300 mL) and H.sub.2O (50
mL). The organic layer was dried, filtered, and concentrated to get
a brown oil. Isolation by preparative HPLC gave compound 12 as a
white solid (85 mg, 48%). .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 0.34-0.46 (m, 2H), 0.48-0.59 (m, 2H), 2.53-2.57 (m,
1H), 3.73 (d, J=11.12 Hz, 1H), 3.96 (dd, J=13.14, 2.02 Hz, 1H),
4.26-4.50 (m, 6H), 6.37 (br. s., 1H), 6.78 (s, 2H), 7.27 (d, J=1.77
Hz, 1H), 7.33-7.35 (m, 1H), 7.38 (s, 1H), 7.48-7.55 (m, 1H). LCMS
(M+H).sup.+: 552.
##STR00047##
Compound 12a
2-amino-4-[2,4-dichloro-6-(2-chloro-ethoxy)-phenyl]-5,7-dihydro-pyrrolo[3,-
4-d]pyrimidine-6-carboxylic acid cyclopropylamide
[0249] 1-Bromo-2-chloroethane (0.4 mL, 4.2 mmol) was added to a
solution of compound 2a (800 mg, 2.1 mmol) and potassium carbonate
(872 mg, 6.3 mmol) in DMF (8 mL). The resulting mixture was heated
at 50.degree. C. for 12 h. The reaction mixture was filtered off to
remove carbonate salt and washed well with EtOAc. The filtrate was
then partitioned between EtOAc (200 ml) and sat. NaHCO.sub.3 (50
ml) and brine (50 ml). The organic layers were dried
(Na.sub.2SO.sub.4), then concentrated by vacuum. This oil residue
was purified by silica gel chromatography (gradient elution
0.fwdarw.10% CH.sub.3OH in dichloromethane) to afford compound 12a
(786 mg, 85%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) d ppm 0.32-0.43
(m, 2H), 0.47-0.58 (m, 2H), 3.69-3.86 (m, 1H), 4.00-4.12 (m, 2H),
4.19 (d, J=13.14 Hz, 2H), 4.23-4.31 (m, 2H), 4.32-4.45 (m, 2H),
6.42 (d, J=2.78 Hz, 1H), 6.80 (s, 2H), 7.38 (d, J=1.77 Hz, 1H),
7.96 (s, 1H). LCMS (M+H).sup.+ 444.
##STR00048##
Examples 13-16 were prepared using methods similar to those
described in the above examples.
TABLE-US-00001 TABLE 1 Ex Name, analytical data and # Structure
synthetic method 13 ##STR00049## 2-amino-N-cyclopropyl-4-(2,4-
dichloro-6-{2-[3-(trifluoromethyl)-1H-
pyrazol-1-yl]ethoxy}phenyl)-5,7-
dihydro-6H-pyrrolo[3,4-d]pyrimidine- 6-carboxamide .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. ppm 0.31-0.42 (m, 2 H), 0.47-0.56 (m, 2
H), 2.48-2.55 (m, 1 H), 3.65 (d, J = 13.14 Hz, 1 H), 3.97 (d, J =
13.14 Hz, 1 H), 4.30-4.37 (m, 2 H), 4.40 (br. s., 2 H), 4.46 (t, J
= 4.67 Hz, 2 H), 6.34 (br. s., 1 H), 6.45 (d, J = 2.02 Hz, 1 H),
6.81 (s, 2 H), 7.26 (d, J = 1.77 Hz, 1 H), 7.30-7.38 (m, 2 H)
Method of Example 12 14 ##STR00050## 2-amino-N-cyclopropyl-4-(2,4-
dichloro-6-{2-[5-(trifluoromethyl)-1H-
pyrazol-1-yl]ethoxy}phenyl)-5,7-
dihydro-6H-pyrrolo[3,4-d]pyrimidine- 6-carboxamide .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. ppm 0.35-0.47 (m, 2 H), 0.55 (d, J =
7.07 Hz, 2 H), 2.52- 2.59 (m, 1 H), 3.20 (d, J = 13.14 Hz, 1 H),
3.78 (d, J = 12.88 Hz, 1 H), 4.30 (s, 2 H), 4.34-4.54 (m, 4 H),
6.31 (br. s., 1 H), 6.55 (d, J = 1.77 Hz, 1 H), 6.70 (s, 2 H), 7.32
(s, 2 H), 7.45 (d, J = 1.52 Hz, 1 H) Method of example 12 15
##STR00051## 2-amino-N-cyclopropyl-4-(2,4-
dichloro-6-{2-[3-methyl-5- (trifluoromethyl)-1H-pyrazol-1-
yl]ethoxy}phenyl)-5,7-dihydro-6H- pyrrolo[3,4-d]pyrimidine-
6-carboxamide .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm
0.28-0.42 (m, 2 H), 0.51 (d, J = 7.07 Hz, 2 H), 1.78 (s, 3 H),
2.52-2.56 (m, 1 H), 3.43 (br. s., 1 H), 3.90 (d, J = 13.14 Hz, 1
H), 4.20-4.37 (m, 3 H), 4.36-4.48 (m, 3 H), 6.22 (s, 1 H), 6.29 (s,
1 H), 6.82 (s, 2 H), 7.28 (d, J = 1.77 Hz, 1 H), 7.34 (d, J = 1.77
Hz, 1 H) Method of example 12. 16 ##STR00052##
2-amino-N-cyclopropyl-4-(2,4- dichloro-6-{2-[5-methyl-3-
(trifluoromethyl)-1H-pyrazol-1- yl]ethoxy}phenyl)-5,7-dihydro-6H-
pyrrolo[3,4-d]pyrimidine- 6-carboxamide .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 0.35 (br. s., 2 H), 0.47 (br. s., 2 H),
2.06 (s, 3 H), 2.45- 2.50 (m, 1 H), 3.10 (d, J = 1.01 Hz, 1 H),
3.72 (d, J = 2.02 Hz, 1 H), 4.24 (d, J = 20.72 Hz, 3 H), 4.34 (br.
s., 3 H), 6.20-6.33 (m, 2 H), 6.64 (s, 2 H), 7.18-7.29 (m, 2 H)
Method of example 12
HSP-90 Biochemical Assay
[0250] Compounds of the present invention were evaluated for
potency against HSP-90 using a SPA (scintillation proximity assay)
competition binding assay. Either full length or N-terminal HSP-90
that contains a 6-His tag on its C-terminus binds to copper on
Yttrium-silicate scintillant beads via the His-tag. Tritiated
propyl-Geldanamycin (pGA), whose structure is shown below, is an
analog of a natural inhibitor of HSP-90 called Geldanamycin.
Tritiated pGA, which contains a tritiated propyl-amine group added
at the #17 position, binds HSP-90 and brings the isotope into
proximity with the beads. 17-n-propylamino-Geldanamycin can be
prepared as described in U.S. Pat. No. 4,261,989, which is
incorporated herein by reference. A second tritiated compound that
can also be used in this assay is shown below and is designated as
Compound A.
##STR00053##
The "T" in the structure of Compound A above indicates the position
of the labeled tritiated hydrogen atoms. This compound has a
K.sub.d of 40 nM and can be prepared as follows. Compound A can be
prepare from the parent compound of Compound A,
(N-allyl-2-(5-chloro-2,4-dihydroxybenzoyl)isoindoline-1-carboxamide)
as described in the following. Allylamine (2.5 mL, 5 mmol, 2M in
THF) was added to a solution of Boc(R,S)-1,3-dihydro-2H-isoindole
carboxylic acid (263 mg, 1 mmole), diisopropylethyl amine (0.9 mL,
5 mmol), and
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium phosphorus
pentafloride (HATU) (420 mg, 1.1 mmol) in 5 mL of DMF under a
nitrogen atmosphere. The reaction was allowed to stir at room
temperature for 12 hours. Saturated NaHCO.sub.3 (30 mL) was added
to the reaction mixture to quench the reaction. EtOAc (2.times.50
mL) was then added to extract the aqueous solution. Dry EtOAc layer
over Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 was filtered off and
the filtrate was evaporated to give a brown oil residue. The
residue was purified by silica gel chromatography (gradient elution
40.fwdarw.50% EtOAc in hexanes) to give the desired intermediate
product (321 mg, quantitative yield) tert-butyl
1-[(allylamino)carbonyl]-1,3-dihydro-2H-isoindole-2-carboxylate.
[0251] Hydrogen chloride (3 mL, 12 mmol; 4 M in dioxane) was added
to a solution of tert-butyl
1-[(allylamino)carbonyl]-1,3-dihydro-2H-isoindole-2-carboxylate (1
mmol) in DCM (5 mL) at room temperature. The reaction was heated
and stirred at room temperature for 12 hours. The reaction mixture
was evaporated to give an oil residue. The residue
(N-allylisoindoline-1-carboxamide) was used for the next step
reaction without further purification.
[0252] N-allylisoindoline-1-carboxamide (1 mmol) was then added to
a solution of 5-chloro-2,4-bis(methoxymethoxy)benzoic acid (which
can be prepared as shown in WO 2006/117669) (340 mg, 1.2 mmol),
4-methylmorpholine (2.2 mL, 20 mmol),
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (460
mg, 2.4 mmol), and 1-hydroxy benzotriazole (330 mg, 2.4 mmol) in 12
mL of DMF under a nitrogen atmosphere. The reaction was allowed to
stir at room temperature for 12 hours. H.sub.2O (50 mL) was added
to the reaction mixture to quench the reaction. EtOAc (2.times.100
mL) was then added to extract the aqueous solution. Dry EtOAc layer
over Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 was filtered off and
the filtrate was evaporated to give a brown oil residue. The
residue was purified by silica gel chromatography (gradient elution
50.fwdarw.60% EtOAc in hexanes) to give the desired intermediate
product (423 mg, 91.8% yield)
N-allyl-2-[5-chloro-2,4-bis(methoxymethoxy)benzoyl]isoindoline-1-carboxam-
ide.
[0253] Hydrogen chloride (4 mL, 16 mmol; 4 M in dioxane) was added
to a solution of
N-allyl-2-[5-chloro-2,4-bis(methoxymethoxy)benzoyl]isoindoline-1-carboxam-
ide (392 mg, 0.85 mmol) in DCM (5 mL). The reaction was stirred at
room temperature for 12 hours. The reaction mixture was neutralized
with saturated NaHCO.sub.3 (aq) and then extracted with EtOAc
(2.times.50 mL). The combined organic layers were dried, filtered,
and evaporated to give the desired final product as the parent
compound
(N-allyl-2-(5-chloro-2,4-dihydroxybenzoyl)isoindoline-1-carboxamide)
as a white solid (221 mg, 69.7% yield). .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 3.57 (d, J=79.33 Hz, 2H), 4.65-4.93 (m,
1H), 4.97-5.19 (m, 1H), 5.42-5.70 (m, 1H), 5.68-5.95 (m, 1H),
6.40-6.71 (m, 1H), 6.92 (s, 1H), 7.15-7.67 (m, 4H), 8.28 (s, 1H),
10.06 (s, 1H), 10.40 (s, 1H). Anal. Calcd for
C.sub.19H.sub.17ClN.sub.2O.sub.4: C, 61.21; H, 4.60; N, 7.51.
Found: C, 61.02; H, 4.63; N, 7.36.
[0254] Once the parent compound was made, Compound A was prepared
using standard hydrogenation methods using tritium gas.
[0255] The beta signal emitted from the isotope excites the
scintillant, which creates a measurable signal. As competitive
compounds are added to the assay mixture, they compete with bound
tritiated pGA or Compound A at the ATP-binding site on the
N-terminal of HSP-90. When a compound displaces the labeled pGA or
Compound A, the signal is reduced (the beta-particles are no longer
in proximity with the bead). This reduction in signal is used to
quantify the extent to which the inhibitor/compound is competitive
with pGA or Compound A.
[0256] The SPA assay for .sup.3H-PGA (designated G1) and Compound A
(designated G2) binding to HSP-90 was performed in 96-well flat
bottom white plates (Corning #3604). For G1, typical reaction
solutions contained 30 nM HSP-90 and 200 nM .sup.3H-PGA in binding
buffer (100 mM Hepes, pH 7.5 and 150 mM KCl). For G2, typical
reaction solutions contained 5 nM HSP-90 and 50 nM of Compound A.
For G1, the .sup.3H-PGA was first diluted to 33% label with
unlabeled pGA that was synthesized and purified to give a final
concentration of 200 nM. For G2, labeled Compound A was diluted
with unlabeled Compound A to provide a ratio of labeled:unlabeled
of 1:2 for a final concentration of 50 nM. Inhibitors were added to
the HSP-90/.sup.3H-PGA (or HSP-90/Compound A) solutions at eleven
different concentrations for K.sub.i determinations. The range of
inhibitor concentrations were 100 .mu.M, or an appropriate range,
for solid samples and 10 .mu.M for targeted library compounds and 4
mM liquid stocks. To determine percent inhibition, the compound was
tested at 1 and 10 .mu.M. The final DMSO in the samples was 4%.
Copper-Ysi beads (Amersham, #RPNQ0096) that have been diluted in
binding buffer were added to each well to give a final
concentration of 100 .mu.g/well. The plates were sealed, covered
with a foil-covered 10d and shaken for 30 minutes at room
temperature. The beads were allowed to settle for 30 minutes after
which the plates were counted using a Packard TopCount NXT
instrument. This procedure has also been adapted for medium
throughput using a Beckman Biomek FX. Samples were run in duplicate
and on two separate days to assure an accurate value of
K.sub.i.
[0257] For K.sub.i determinations, the corrected cpm's (actual
cpm's minus background) were plotted vs. inhibitor concentration
using GraphPad Prism software. The data were fit to a generic
IC.sub.50 equation, Y=YI/(1+[X]/IC.sub.50), where YI=Y-intercept
and [X] is the competing ligand/inhibitor. The IC.sub.50 was then
used to calculate the Ki by using the Cheng-Prusoff equation:
Ki { cl } = IC 50 { cl } 1 + ( [ hl ] / Kd { hl } )
##EQU00001##
Where cl=cold ligand concentration (varies), [hl]=concentration of
hot ligand (200 nM or 50 nM) and Kd{hl}=240 nM (for .sup.3H-PGA) or
40 nM (for Compound A). Error was calculated as follows: IC.sub.50
error/IC.sub.50 value=fractional error and fractional error*K.sub.i
value=K.sub.i error.
[0258] In the cases in which inhibitor binds to HSP-90 so tightly
that the population of free inhibitor molecules is significantly
depleted by formation of the enzyme-inhibitor complex, the above
equation is no longer valid. This is normally true when the
observed IC.sub.50 is about the same as the HSP-90 concentration.
For a tight binding inhibitor, the following equation can be
applied:
EL EL o = - ( K I app + I o - E o ) + ( K I app + I o - E o ) 2 + 4
.times. E o .times. K I app 2 .times. E o ##EQU00002## where K I
app = K I .times. ( 1 + L o K L ) ##EQU00002.2##
EL and EL.sub.o are the radioligand-HSP-90 complexes in the
presence and absence of inhibitor, respectively. EL/EL.sub.o
represents the fractional signal in the presence of inhibitor. Io,
E.sub.o, and L.sub.o are the inhibitor, HSP-90, and radioligand
concentrations, respectively. K.sub.I is the inhibition constant
for the ligand, while K.sub.L is the binding affinity constant
between the enzyme (HSP-90) and the ligand.
[0259] The Ki assay data of the compound 1-16 of Examples 1-16 are
listed in the following Table 2.
TABLE-US-00002 TABLE 2 EX# (G2): Ki (nM) Akt Lum: IC.sub.50 (.mu.M)
1 4.68 0.0384 2 3.38 0.104 3 3.39 0.0525 4 2.05 0.0164 5 1.69
0.0702 6 3.32 0.0328 7 3.44 0.0209 8 1.50 0.0370 9 6.27 0.0357 10
2.86 0.111 11 64.3 2.55 12 4.69 0.175 13 7.76 0.660 14 75.1 6.69 15
437 18.3 16 259 9.96
* * * * *